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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INimNA'nONAL APPLICATIONT>UBUSHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :

C12N 15/12, 15/62, C07K 14/47, 16/18,

G01N 33/53

(11) International Publication Number: WO 95727059

(43) International Publication Date: 12 October 1995 (12.10.95)

(21) International Application Number: PCT/US95/04O75

(22) International Filing Date: 31 March 1995 (31.03.95)

(30) Priority Data:
08/222,619

31 March 1994 (31.03.94)

(71) Applicants: AMGEN INC. [US/US]; Amgen Center, 1840

Dehavilland Drive, Thousand Oaks, CA 91320-1789 (US).
TOE ROCKEFELLER UNIVERSITY [US/US]; 1230 York
Avenue, New York, NY 10021-6399 (US).

(72) Inventors: LICHENSTEIN, Henri, Stephen; 9586 Lucerne

Street, Ventura, CA 93004 (US). LYONS, David, Edwin;
2027 Truett Circle, Thousand Oaks, CA 91320-1789 (US).
WURFEL, Mark, Matsuo; 420 E. 70 Street, New York, NY
10021 (US). WRIGHT, Samuel, Donald; 2 Briar Close,
Larchmont, NY 10538 (US).

(74) Agents: ODRE, Steven, M. et al.; Amgen Inc., Amgen Center,
1840 Dehavilland Drive, Thousand Oaks, CA 91320-1789
(US).

(81) Designated States: AM, AT, AU, BB, BG, BR, BY, CA, CH,
CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, JP, KE, KG,
KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, MN, MW,
MX, NL, NO, NZ, PL, FT, RO, RU, SD, SE, SG, SI, SK,
TJ, TM, TT, UA, UG, UZ, VN, European patent (AT, BE,
CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, FT,
SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML,
MR, NE, SN, TO, TG), ARIPO patent (KE, MW, SD, SZ,
UG).

Published

With international search report

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of
amendments.

(54) Title: AFAMIN: A HUMAN SERUM ALBUMIN-LIKE PROTEIN

(57) Abstract

The invention relates to a novel human serum protein referred to as AFM, which has one or more activities in common with human
serum albumin, human a-fetoprotein, or human vitamin D binding protein and which has an apparent molecular weight by SDS-PAGE of
87 kd; variants thereof; and related genes, vectors, cells and methods.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Austria

United Kingdom

Mauritania

Australia

Georgia

Malawi

Barbados

Guinea

Niger

Belgium

Greece

Netherlands

Burkina Faso

Hungary

Norway

Bulgaria

Ireland

New Zealand

Benin

Italy

Poland

Brazil

Japan

Portugal

Belarus

Kenya

Romania

Canada

Kyrgystan

Russian Federation

Central African Republic

Democratic People's Republic

Sudan

Congo

of Korea

Sweden

Switzerland

Republic of Korea

Slovenia

Cote d'lvoire

Kazakhstan

Slovakia

Cameroon

Liechtenstein

Senegal

China

Sri Lanka

Chad

Czechoslovakia

Luxembourg

Togo

Czech Republic

Latvia

Tajikistan

Germany

Monaco

Trinidad and Tobago

Denmark

Republic of Moldova

Ukraine

Spain

Madagascar

United States of America

Finland

Mali

Uzbekistan

France

Mongolia

Viet Nam

Gabon

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AFAMIN: A HUMAN SERUM ALBUMIN-LIKE PROTEIN

Fieia of the invention

Generally, the invention relates to the field
of human serum proteins that are functionally and ■
structurally similar to the related proteins: human
serum albumin (ALB) , human a- fetoprotein (AFP), and
10 vitamin D-binding protein (VDB) .

Background of the Invention

The human serum proteins albumin (ALB) ,
15 a -feto-protein (AFP) and vitamin D binding protein (VDB)

are known to be members of a multigene ALB family. All
three proteins are found in serum where they mediate the
transport of a wide variety of ligands . ALB binds fatty
acids, amino acids, steroids, glutathione, metals,

20 bilirubin, lysolecithin, hematin, prostaglandins and
pharmaceuticals (for review, see 1) . AFP binds fatty
acids, bilirubin and metals (2, 3) . VDB binds vitamin D
and its metabolites as well as fatty acids, actin, C5a
and C5a des Arg. (4-7) •

25 In addition to their transport capabilities,

ALB family proteins possess a wide assortment of other
functional activities. ALB is the main contributor to
the colloid oncotic pressure of plasma, acts as a
scavenger of oxygen-free radicals and can inhibit

30 copper-stimulated lipid peroxidation, hydrogen peroxide
release, and neutrophil spreading (1, 8-10) . AFP has
been implicated in the regulation of immune processes
(11-14) and VDB can act as a co-chemotactic factor for
neutrophils (6, 15) and as an activating factor for

35 macrophages (16) .

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The serum levels of ALB family proteins are
also known to be responsive to various pathological
conditions. ALB is a negative acute phase protein (17)
whose levels decrease in times of stress. AFP levels are
5 elevated in women carrying fetuses with certain

developmental disorders (18, 19) and in individuals with
hepatocarcinoma, teratocarcinoma, hereditary tyrosinemia
or ataxia-telangiectasia (20-24) . VDB levels are
decreased in patients with septic shock (25) or

10 fulminant hepatic necrosis (26, 27) .

ALB family members also have significant
structural similarities. Homology has been observed at
the primary amino acid sequence level and there is also
a well-conserved pattern of Cys residues which predicts

15 similar secondary structures (28-32) . ALB family genes
have similar exon/intron organizations (33-36) and all
have been mapped to human chromosome 4„ within the region
4qll-q22 (37; 38) .

Human "Afamin" (abbreviated as "AFM") is a

20 novel serum protein with a molecular weight of 87000

daltons. It shares strong similarity to albumin- family
members and has the characteristic pattern of disulfide
bonds observed in this family. In addition, the gene
maps to chromosome 4 as do other members of the albumin

25 gene family. Thus, AFM is the fourth member of the
albumin family of proteins . AFM cDNA was stably
transfected into Chinese hamster ovary cells and
recombinant protein (rAFM) was purified from conditioned
medium. Both rAFM and AFM purified from human serum

30 react with a polyclonal antibody that was raised against
a synthetic peptide derived from the deduced amino acid
sequence of AFM. It is expected that AFM will have
properties and biological activities in common with ALB,
AFP, and VDB.

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Pnhlirations relating to "Background of the Invention"

1. Peters, T. Jr., Adv. Protein Chem. 37, 161 - 245
(1985) .

2. - Parmelee, D.C., Evenson, M.A., and Deutsch, H.F.. J.
Biol. Chem. 253, 2114 - 2119 (1978).

3. Berde, C.B., Nagai, M. , Deutsch, H.F., J. Biol.
10 Chem. 254, 12609 - 12614 (1979) .

4. Daiger, S.P., Schanfield, M.S., and Cavalli-Sforza,
L.L., Proc. Nat. Acad. Sci. U.S.A. 72, 2076 t 2080
(1975) .

5. Van Baelen, H., Bouillon, R. , and De Moor, P., J".
Biol. Chem. 255, 2270 - 2272 (1980).

6. Kew, R.R., and Webster, R.O., J. Clin. Invest. 82,
20 364 - 369 (1988) .

7. Williams, M.H., Van Alstyne, E.L., and Galbraith,
R.M., Biochem. Bipophys. Res. Commun. 153, 1019 - 1024
(1988) .

8. Holt, M.E., Ryall, M.E.T., and Campbell, A.K., Br.
J. exp. Path. 65, 231 - 241 (1948) .

9. Gutteridge, J.M.C., Biochim. Biophys ACTA 869, 119
30 -127 (1986) .

10. Nathan, C, Xie, Q-W. , Halbwachs-Mecarelli, L. and
Jin, W.W., J. Cell Biol. 122, 243-256 (1993).

35 11. Yachnin, S., Proc. Natl. Acad. Sci. U.S.A. 73, 2857
- 2861 (1976) .

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12. Auer, I.O., and Kress, H.G., Cell. Immunol. 30, 173

- 179 (1977) .

5 13, Alpert, E., Dienstag, J.L., Sepersky, S., Littman f
B., -and Rocklin, R., Immunol . Commun . 7, 163 - 185
(1978) .

14, Chakraborty, M., and Mandal, C, Immunol. Invest.
10 22, 329 - 339 (1993) .

15. Perez, H.D., Kelly, E., Chenoweth, D., and Elf man,
F . , J. Clin. Invest. 82, 360 - 363 (1988).

15 16. Yamamoto, N. and Homma, S., Proc. Natl. Acad. Sci.
U.S.A. 88, 8539 - 8543 (1991).

17. Koj, A., in The Acute Phase Response to Injury and
Infection 1 (Gordon, A.H., and Koj, A. eds) pp. 45 - 160 '

20 (1985) .

18. Brock, D.J.H., and Sutcliffe, R.G., Lancet 2, 197

- 199 (1972) .

25 19. Allan, L.D., Ferguson-Smith, M.A., Donald, I.,
Sweet, E.M. and Gibson, A. A.M., Lancet 2 522 - 525
(1973) .

20. Waldmann, T.A., and Mclntire, K.R., Lancet 2, 1112
30 - 1115 (1972) .

21. Belanger, L., Belanger, M. , Prive, L., Larochelle,
J., Tremblay, M. , and Aubin, G., Pathol. Biol. 21, 449 -
455 (1973).

22. Belanger, L., Pathol. Biol. 21, 457 - 463 (1973).

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23. Ruoslahti, E . , Pihto, H., and Seppala, M. ,
Transplant. Rev. 20, 38 - 60 (1974).

5 24. Tamaoki, T . , and Fausto, in Recombinant DNA and
Cell Proliferation (Stein G. and Stein J. eds.) pp. 145
- 168 (1984) .

25. Lee, W.M., Reines, D., Watt, G.H., Cook, J. A. ,

10 Wise, W.C., Halushka, P.V., and Galbraith, R.M., Circ.
Shock. 28, 249 - 255 (1989).

26. Young, W.O., Golds chmidt-Clermont, P. J., Emerson,
D.L., Lee, W.M., Jollow, D . J . , and Galbraith, R.M., Lab.

15 Clin. Med. 110, 83 - 90 (1987).

27. Golds chmidt-Clermont, P.J., Lee, W.M., and
Galbraith, R.M., Gastroenterology 94, 1454 - 1458
(1988) .

28. Lawn, R.M., Adelman, J. , . Bock, S.C., Franke, A.E.,
Houck, CM. , Najarian, R.C., Seeburg, P.H., and Wion,
K.L., Nucleic Acid Res. 9, 6103 - 6114 (1981).

25 29. Dugaiczyk, A., Law, S.W., and Dennison, O.E., Proc.
Natl. Acad. Sci. U.S.A. 79, 71 - 75 (1982).

30. Morinaga, T . , Sakai, M. , Wegmann, T.G., and
Tamaoki, T., Proc. Natl. Acad. Sci. U.S.A. 80, 4604 -

30 4608 (1983) .

31. Cooke, N.E. and David, E.V., J. Clin. Invest. 76,
2420 - 2424 (1985) .

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32. Yang, F., Brune, J.L., Naylor, S.L., Cupples, R.L.,
Naberhaus, K.H. , and Bowman, B.H., Proc. Natl. Acad.
Sci. U.S.A. 82, 7994 - 7998 (1985).

5 33. Sakai, M. , Morinaga, T., Urano, Y. , Watanabe, K.,
Wegmann, T.G., and Tamaoki, T., J*. Biol. Chem. 260, 5055
- 5060 (1985) .

34. Minghetti, P.P., Ruffner, D.E., Kuang, W-J.,
10 Dennison, O.E., Hawkins, J.W., Beattie, W.G., and

Dugiaczyk, A., J. Biol. Chem. 261, 6747 - 6757 (1986).

35. Gibbs, P.E.M., Zielinski, R. , Boyd, C, and
Dugaiczyk, A., Biochemistry 26, 1332 - 1343 (1987).

36. Witke, W.F., Gibbs, P.E.M., Zielinski, R., Yang,

F . , Bowman, B.H., and Dugaiczyk, A., Genomics 16, 751 -
754 (1993) .

20 37. Mikkelsen, M. , Jacobsen, P. and Henningsen, K. ,
Hum. Hered. 27, 105 - 107 (1977) .

38. Harper, M.E., and Dugaiczyk, A., Am. J. Hum. Genet.
35, 565-572 (1983) .

The sections below contain a summary of
background information that is currently available on
ALB, AFP, and VDB and contains lists of additional
publications relating to these known proteins.

I. Human Serum Albumin

Human serum albumin is an important factor in
the regulation of plasma volume and tissue fluid balance
35 through its contribution to the colloid osmotic pressure
of plasma. Albumin normally constitutes 50-60% of

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plasma proteins and because of its relatively low
molecular weight (66,300-69,000), exerts 80-85% of the
colloidal osmotic pressure of the blood.

The best known functions of ALB involve
5 regulation of transvascular fluid flux and hence, intra
and extravascular fluid volumes and transport of lipid
and lipid-soluble substances. ALB solutions are •
frequently used for plasma volume expansion ' and
maintenance of cardiac output in the treatment of

10 certain types of shock or impending shock including
those resulting from burns, surgery, hemorrhage, or
other trauma or conditions in which a circulatory volume
deficit is present. Transfusions of whole blood or red
blood cells also may be necessary, depending on the

15 severity of red blood cell loss.

Intravenous (IV) administration of
concentrated ALB solutions causes a shift of fluid from
the interstitial spaces into the circulation and a
slight increase in the concentration of plasma proteins.

20 When administered IV to a well-hydrated patient, each
volume of 25% ALB solution draws about 3.5 volumes of
additional fluid into the circulation within 15 minutes,
reducing hemoconcentration and blood viscosity. In
patients with reduced circulating blood volumes (as from

25 hemorrhage or loss of fluid through exudates or into
extravascular spaces), hemodilution persists for many
hours, but in patients with normal blood volume, excess
fluid and protein are lost from the circulation ■ within a
few hours. In dehydrated patients, ALB generally

30 produces little or no clinical improvement unless
additional fluids are administered.

Although ALB contains some bound amino acids,
it provides only modest nutritive effect. ALB binds and
functions as a carrier of intermediate metabolites

35 (including bilirubin), trace metals, some drugs, dyes,
fatty acids, hormones, and enzymes, thus affecting the

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transport , inactivation, and/or exchange of tissue
products .

ALB is also involved in a number of other
vital functions, some of which have only recently been
5 suggested and perhaps others which are as yet

unrecognized. Among recognized unique features of
albumin are: a) binding, and hence, inactivation of
toxic products; b) regulation of the plasma and
interstitial fluid concentrations of endogenous and

10 exogenously administered substances and drugs; c)
involvement in anticoagulation; d) maintenance of
microvascular permeability to protein; and e) scavenging
of free radicals and prevention of lipid peroxidation.
This latter property may prove to be critically

15 important, particularly in inflammatory disease states

in which free radicals are thought to be a major culprit
in direct damage due to tissue oxidation, and indirect
tissue damage due to inactivation of important
antiproteinases such as ai~PI and AT-III.

The following is a more detailed summary of
the many uses for ALB that have been reported in the
literature :

25 a. Functions pf ALB

• Contributes to colloid osmotic pressure
and thus prevents water loss from circulation;

30 • Aids in transport, distribution,

metabolism of fatty acids (primarily long chain) , amino
acids (Cys and Trp) , steroids, glutathione, metals (Ca,
Zn) , bilirubin, lysolecithin, hematin, prostaglandins
and pharmaceuticals to liver, intestine, kidney and

35 brain presumably through specific albumin receptors that
have been identified on the endothelium;

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Serves as a reservoir for fatty acids
intra and extravascularly (60% of the ALB is found
extravascularly) ;

• Modification of doxorubicin (DXR) by
conjugating it to bovine serum albumin (BSA) improved
chemotherapeutic efficiency of DXR presumably by
decreasing efflux of BSA-DXR compared to DXR alone (in

10 animal models), suggesting a similar use with ALB;

• Inhibits Cu-stimulated lipid peroxidation
and hemolysis of erythrocyte membranes (acts as
antioxidant) ;

Scavenges HOC1 and peroxy radicals;

Prevents peroxidation of fatty acids by
binding to them;

May exert a protective effect in body
fluids that have little endogenous antioxidant
protection (e.g., eye and cerebrospinal fluids) ;

25 • In urine, high levels of ALB are

diagnostic for detection of early renal pathology in
diabetics;

• Administered to combat shock and given to
30 neonates with respiratory distress syndrome;

Administered as a vehicle for hematin to
treat acute intermittent porphyria;

35 • Used in tissue culture in place of whole

serum;

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Enhances effectiveness of superoxide
dismutase (SOD) when coupled to SOD through enhanced
serum half -life;

♦ In microsphere form, ALB is useful as a
carrier of therapeutic agents;

• Inhibits hydrogen peroxide release and
10 neutrophil spreading.

B. publications relating tQ ALB

15 38. Peters, Theodore in ALBUMIN An Overview and
Bibliography, Second Edition, 1992.

39. American" Hospital Formulary Service Drug
Information, Blood Derivatives, 762-763 (1992) .

40. Yamashita, T. , etal., Biochem. Biophys. Res.
Commun. 191 (2), 715-720 (1993).

41. Candlish, John K., Pathology 25, 148-151 (1993).

42. Ohkawa, K., et al., Cancer Research 54, 4238-4242
(1993) .

43. He, Xiao Min and Carter, Daniel C, Nature 358,
30 209-215 (1992) .

44. Brown, J. M. , et al . , . Inflammation 13 (5), 583-589
(1989) .

35 45. Emerson, T. E., Critical Care Medicine 17 (7), 690-
694 (1989) .

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46. Halliwell, Barry, Biochem. Pharmacol. 37 (4), 569-
571 (1988) .

5 47. Holt, M. E . , et al., Br. J". Exp. Path. 65, 231-241
(1984).

II. Alpha Fetoprotein

10 Alpha-f etoprotein (AFP; molecular weight

70,000) is a major serum protein produced during
development and is produced primarily by the fetal liver
and yolk sac cells. Its synthesis decreases markedly
after birth and only trace amounts are present in the

15 serum of adults. Increased adult serum levels are a

sign of hepatoma or yolk sac tumor, since these tumors
produce AFP. The specific associations of AFP with
fetal development as well as the above type of
malignancies has attracted much interest and many

20 studies have been done on the structure of AFP and its
gene, the regulation of gene expression, and biological
functions .

Similar to ALB, AFP has been shown to bind
various ligands such as unsaturated fatty acids,
25 estrogens, bilirubin, copper and' nickel ions, and

others . AFP also has been claimed to regulate immune
processes in a variety of systems from many different
laboratories, although the results are controversial.

The following is a more detailed list of uses
30 for AFP that are available in the literature:

a. Functions of AFP

• Binds unsaturated fatty acids, estrogens,
35 bilirubin, Cu, Ni;

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Elevated levels in amniotic fluid of
pregnant women indicative of fetal malformations;

• High levels also found in hereditary
5 tyrosinemia and ataxia-telangiectasia (autosomal

recessive disorder characterized by a defect in tissue
differentiation of thymus and liver) ;

Inhibits NK cell activity;

• Induces T suppressor cells;

• Inhibits mitogenic responses of
lymphocytes to PHA and ConA;

Inhibits T cell proliferation to la

determinants ;

• Decreases macrophage phagocytosis and la
20 expression;

Inhibits FSH-mediated estradiol
production by porcine granulosa cells;

25 • Enhances growth-factor mediated cell

proliferation of porcine granulosa cells.

B. Publications relating to AFP

30 48. Suzuki, Y., et al., J". Clin. Invest. 90,

1530-1536 (1992) .

49. Sakai, M., et al., J. Biol. Chem. 260
(8) , 5055-5060 (1985) .

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50. EPO Patent Application No. 0353814,
February 7, 1990.

in. vitemin-p Binding Protein

The group-specific component (Gc; VDB) is an
a2~globulin of molecular weight 51,000. It is
synthesized in the liver and is the major vitamin D-
binding protein in plasma. VDB appears in human

10 populations as three common genetic phenotypes: Gel,

Gc2, and Gc2-1. VDB has also been reported to bind G-
actin and to be spatially associated with IgG on
lymphocyte membranes .

The following is a more detailed list of uses

15 for VDB that are available in the literature:

A. Functions of VDB

• Binds seco-steroid, vitamin D and the
20 derivatives 25-hydroxy vitamin D and 1,25 hydroxy

vitamin D, possibly for transport in plasma;

• 1,25 vitamin D can differentiate
monocytes and VDB prevents this;

• Binds actin (prevents assembly of actin

polymers) ;

Binds unsaturated fatty acids (e.g.,
30 arachidonic acid) ;

• Binds C5a and C5a des Arg to act as a
cochemotactic factor for neutrophils;

35 • Acts as an activating factor for

macrophages.

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B. Publication relating to VDB

51. Watt, G. H., et al., Circulatory Shock
5 28, 279-291 (1989) .

The protein of the present invention, AFM,
bears a strong similarity in structure to ALB, AFP, and
VDB, and is therefore expected to share the above
10 utilities and activities with the known proteins
discussed above.

Summary of the Invention

15 In the course of experiments designed to

purify a serum protein which could inhibit the binding
of lipopolysaccharide (LPS) -coated erythrocytes to human
macrophages, the inventors purified a novel human
protein that co-purifies with apolipoprotein Al (ApoAl) . "

20 The novel protein has an apparent molecular weight of
87,000 when run on SDS-PAGE and is designated as AFM.
Herein, the inventors describe the cloning of the cDNA
for AFM and demonstrate that AFM has a striking
similarity, both structurally and functionally/ to other

25 members of the ALB family. In addition, the inventors
purified AFM from the serum-free conditioned medium of
CHO D" cells transfected with the cDNA for AFM,- thus
allowing the study of AFM in the absence of ApoAl .

Based on the above, the present invention

30 provides purified and isolated polynucleotides (e.g.,
DNA sequences and RNA transcripts thereof) encoding a
novel human polypeptide, "AFM" as well as complexes of
AFM with ApoAl and/or lipids, and polypeptide variants
(including fragments and analogs) thereof which display

35 one or more biological activities or properties specific
to AFM.

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Preferred DNA sequences of the invention
include genomic and cDNA sequences as well as wholly or
partially chemically and or enzymatically synthesized
DNA sequences and biological replicas thereof. Also
5 provided are autonomously replicating recombinant
constructions such as plasmid and viral DNA vectors
incorporating such sequences and especially vectors,
wherein DNA encoding AFM or an AFM variant are
operatively linked to an endogenous or exogenous

10 expression control DNA sequence.

According to another aspect of the invention,
host cells, especially unicellular host cells such as
prokaryotic and eukaryotic cells, are stably transformed
with DNA sequences of the invention in a manner allowing

15 AFM and variants thereof to be expressed therein.

Host cells of the invention are useful in
methods for the large scale production of AFM and AFM
variants wherein the cells are grown in a suitable
culture medium and the desired polypeptide products are

20 isolated from the cells or from the growth medium.

Novel AFM and AFM variant products of the
invention may be obtained as isolates from natural cell
sources, but are preferably produced by recombinant
procedures involving host cells of the invention. The

25 products may be obtained in fully or partially

glycosylated, partially or wholly deglycosylated, or
non-glycosylated forms, depending on the host cell
selected for recombinant production and/or post-
isolation processing. The products may also be bound to

30 other molecules, such as cellularly derived lipids
and/or ApoAl .

Products of the invention include monomeric
and multimeric polypeptides having the sequence of amino
acid residues numbered -21 through 578 as set out in

35 FIG. 1 herein. As explained in detail infra f this

sequence includes a putative signal or leader sequence

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which precedes the "mature" protein sequence and spans
residue -21 (Met) through residue -1 (Thr) followed by
the mature protein spanning residues 1 (Leu) to residue
578 (Asn) . Based on amino acid composition, the
5 calculated molecular weight of the mature protein
lacking glycosylation or other post-translational
modification is approximately 66,576 daltons.

AFM variants of. the invention may comprise
fragments including one or more of the regions specified

10 herein and may also comprise polypeptide analogs wherein
one or more of the specified amino acids is deleted or
replaced: (1) without substantial loss, and preferably
with enhancement, of one or more biological activities
or immunological characteristics specific for AFM; or

15 (2) with specific modulation of a particular

ligand/receptor binding function. Analog polypeptides
including additional amino acid residues (e.g., lysine)
that facilitate multimer formation are also
contemplated.

20 Further comprehended by the present invention

are antibodies (e.g., monoclonal and polyclonal .
antibodies, single chain antibodies, chimeric
antibodies, CDR-grafted antibodies and the like) or
other binding proteins which are specific for AFM or AFM

25 variants. Antibodies can be developed using isolated
natural or recombinant AFM or AFM variants.

The antibodies are useful in complexes for
immunization as well as for purifying polypeptides of
the invention. The antibodies are also useful in

30 modulating (i.e., blocking, inhibiting or stimulating)
ligand/receptor binding reactions involving AFM.

Anti-idiotypic antibodies specific for anti-
AFM antibodies and uses of such anti-idiotypic
antibodies in treatment are also contemplated. Assays

35 for the detection and quantification of AFM on cell
surfaces and in fluids such as serum may involve a

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single antibody or multiple antibodies in a "sandwich"
assay format.

The uses of the DNA and amino acid sequences
of the present invention are varied. For example,
5 knowledge of the sequence of a cDNA for AFM makes
possible the isolation by DNA/ DNA hybridization of
genomic DNA sequences encoding AFM and specifying -AFM
expression control regulatory sequences such as
promoters, operators and the like. DNA/DNA

10 hybridization procedures carried out -with DNA sequences
of the invention and under stringent conditions are
likewise expected to allow the isolation of DNAs
encoding allelic variants of AFM, other structurally
related proteins sharing the biological and/or

15 immunological specificity of AFM, and proteins

homologous to AFM from non-human species (especially
from other mammals) . DNAs of the invention are useful
in DNA/KNA hybridization assays to detect the capacity
of cells to synthesize AFM. A variety of specific uses

20 for AFM are disclosed herein below. These uses are
primarily based on the- known uses of the homologous
albumin type polypeptides discussed above.

Also made available by the invention are anti-
sense polynucleotides (e.g., DNA and RNA) relevant to

25 regulating expression of AFM by those cells which

ordinarily express it. Furthermore, knowledge of the
DNA and amino acid sequences of AFM make possible the
generation by recombinant means of hybrid fusion
proteins characterized by the presence of AFM protein

30 sequences and immunoglobulin heavy chain constant
regions and/or hinge regions . See, Capon, et al.,
Nature, 337: 525-531 (1989); Ashkenazi, et al., P.N.A.S.
(USA), 88: 10535-10539 (1991); and PCT WO 89/02922,
published April 6, 1989.

i
t

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PCI7US95/04075

BRIEF DESCRIPTION OF THE FIGURES

Numerous other aspects and advantages of the
present invention will therefore be apparent upon
5 consideration of the following detailed description
thereof, reference being made to the drawings wherein:

FIG. 1 shows the nucleotide and deduced amino
acid sequence of AFM. The putative signal sequence is
indicated in lower case letters. Asterisks indicate
10 putative sites for N-glycosylation . These are also
represented as SEQ ID NO:l AND SEQ ID NO: 2.

FIG. 2A and 2B show a comparison of ALB family
amino acid sequences. FIG. 2A shows the alignment of ALB
family proteins. Sequences were aligned using the
15 Clustal method in the MegAlign program (DNASTAR) .
Identical amino acid residues are boxed. Consensus
indicates residues identical in all 4 sequences.
Majority indicates 2 or 3 residues identical in all 4
sequences. FIG. 2B shows percent similarity (right of
20 diagonal) and identity (left of diagonal) between ALB
family members. Similarities were determined using the
GCG GAP program. The sequences for the comparison
proteins are also provided as follows: serum albumin,
SEQ ID NO: 3; alpha fetoprotein, SEQ ID NO: 4; and vitamin
25 D binding protein, SEQ ID NO: 5.

FIG. 3 shows the conserved Cys pattern in ALB
family proteins. The mature form of ALB family proteins
are depicted with thin vertical bars representing single
Cys residues and thick vertical lines representing -Cys-
30 Cys- sequences.

FIG. 4 shows the putative disulfide bonding
pattern for AFM. The organization of domains and double
loops are drawn as originally proposed for ALB.

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FIG. 5 shows the expression of rAFM in stably
transfected CHO D" cells. Samples were applied to SDS-
PAGE under reducing conditions followed by
5 electrophoretic transfer to nitrocelluose . After

blocking with skim milk, the membrane was probed with
the AM339 antibody followed by incubation with donkey
rabbit anti-Ig. Immunoreactive proteins were visualized
by chemiluminescence . Lane 1, 80 ml conditioned medium

10 (CM) from CHO D~ cells transfected with AFM cDNA; lane
2 f 80 ml CM from nontransf ected CHO D~ cells; lane 3,
100 ng AFM purified from human plasma. Size markers (in
kDa) are indicated on the left.

FIG. 6 shows purification of rAFM. Samples

15 purified from CHO D" cells transfected with AFM cDNA
were adjusted with an equal volume of 2X sample buffer
(125 mM Tris-HCl, pH 6.8, 4% SDS, 0.,005% bromophenol
blue, 10% glycerol) and analyzed by SDS-PAGE using 4-20%
polyacrylamide gradient gels (Novex) under reducing

20 conditions. The gel was stained with Coomassie Brilliant
Blue. Lanes 1 and 6 r 10 mg Mark-12 molecular weight
markers (Novex, Inc.); lane 2, 50 mg total protein after
addition of ammonium sulfate to concentrated CM
(supernatant was dialyzed against PBS and subsequently

25 loaded onto the gel) ; lane 3, 25 mg Phenyl Sepharose
water eluate; lane 4, 10 mg Q Sepharose-purif ied rAFM;
lane 5, 1 mg of Superdex 200-purified rAFM.

30 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definition of AFM

AFM is defined as a polypeptide having a
qualitative biological activity or property in common
35 with AFM of FIG. 1.

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Included within the scope of AFM as that term
is used herein is the AFM having the amino acid sequence
of AFM as set forth in FIG. 1, SEQ ID NO: 2;
glycosylated, deglycosylated or unglycosylated
5 derivatives of AFM; and lipidated or delipidated forms
of AFM.

Also included within the scope 1 of AFM are AFMs
from any species , including without limitation: human,
mouse, rat, pig, rabbit, monkey, dog, etc. Especially
10 preferred is the human form of AFM.

Variants of AFM

Variants of AFM include homologous amino acid
sequence variants of the sequence of FIG. 1, and

15 homologous in-vitro-generated variants and derivatives
of AFM, which are capable of exhibiting a biological
activity or property in common with AFM of FIG. 1.

"Homologous" is used herein to refer to the
residues in a candidate sequence that are identical with

20 the residues in the sequence of AFM in FIG. 1 after
aligning the sequences • and introducing gaps, if
necessary, to achieve the maximum percent homology.

A biological activity or property of AFM is
defined as either 1) immunological cross-reactivity with

25 at least one epitope of AFM, or 2) the possession of at
least one regulatory or effector function qualitatively
in common with AFM. Examples of those activities may be
found in the section herein describing uses of AFM.

"Immunologically cross-reactive" as used

30 herein means that the candidate polypeptide is capable
of competitively inhibiting the qualitative biological
activity of AFM or an AFM variant having this activity
with polyclonal antisera raised against the known active
analog. Such antisera are prepared in conventional

35 fashion by injecting animals such as goats or rabbits,

for example, subcutaneously with the known active analog

WO 95/27059 X PCT/US95/04075

in complete Freund's adjuvant, followed by booster
intraperitoneal or subcutaneous injection in incomplete
Freund's adjuvant. An example of production of
polyclonal antisera production is presented in the
5 examples section below.

Amino acid sequence variants of AFM are
prepared with various objectives in mind, including
increasing the affinity of AFM for its binding partner,
facilitating the stability, purification and preparation

10 of AFM, and the like.

Amino acid sequence variants of AFM fall into
one or more of three classes: insertional,
substitutional, or deletional variants. These variants
ordinarily are prepared by site specific mutagenesis of

15 nucleotides in the DNA encoding AFM, by which DNA
encoding the variant is obtained, and thereafter
expressing the DNA in recombinant cell culture.
However, variant AFM fragments having up to about 100-
150 amino acid residues are prepared conveniently by in

20 vitro synthesis.

The amino acid sequence variants of AFM are
predetermined variants not found in nature or naturally
occurring alleles. AFM variants typically exhibit the
same qualitative biological activity as the naturally

25 occurring AFM molecule. However, AFM variants and
derivatives that are not capable of binding to their
ligands are useful nonetheless (a) as a reagent- in
diagnostic assays for AFM or antibodies to AFM, • (b) when
insolubilized in accordance with known methods, as

30 agents for purifying anti-AFM antibodies from antisera

or hybridoma culture supernatant s, and (c) as immunogens
for raising antibodies to AFM or as immunoassay kit
components (labeled, as a competitive reagent for the
native AFM or unlabeled as a standard for AFM assay) so

35 long as at least one AFM epitope remains active.

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While the site for introducing an amino acid
sequence variation is predetermined, the mutation per se
need not be predetermined. For example, in order to
optimize the performance of a mutation at a given site,
5 random or saturation mutagenesis (where all 20 possible
residues are inserted) is conducted at the target codon
and the expressed AFM variant is screened for the -
optimal combination of desired activities. Such
screening is within the ordinary skill in the art.

10 Amino acid insertions usually will generally

be on the order of about from 1 to 10 amino acid
residues; substitutions are typically introduced for
single residues; and deletions will generally range
about from 1 to 30 residues. Deletions or insertions

15 preferably are made in adjacent pairs, i.e. a deletion
of 2 residues or insertion of 2 residues. It will be
amply apparent from the following discussion that
substitutions, deletions, insertions or any combination
thereof are introduced or combined to arrive at a final

20 construct.

Insertional amino acid sequence variants of
AFM are those in which one or more amino acid residues
extraneous to AFM are introduced into a predetermined
site in the target AFM and which displace the

25 preexisting residues . Commonly, insertional variants

are fusions of heterologous proteins or polypeptides to
the amino or carboxyl terminus of AFM. Such variants
are referred to as fusions of AFM and a polypeptide
containing a sequence which is other than that which is

30 normally found in AFM at the inserted position. Several
groups of fusions are- contemplated herein.

Immunologically active AFM derivatives and
fusions comprise AFM and a polypeptide containing a non-
AFM epitope, and are within the scope of this invention.

35 The non-AFM epitope is any immunologically competent

polypeptide, i.e., any polypeptide which is capable of

WO 95/27059 ^° PCT/US95/04075

eliciting an immune response in the animal to which the
fusion is to be administered or which is capable of
being bound by an antibody raised against the non-AFM
polypeptide. Typical non-AFM epitopes will be those
5 which are borne by allergens, autoimmune epitopes, or
other potent immunogens or antigens recognized by pre-
existing antibodies in the fusion recipient, including
bacterial polypeptides such as trpLE, beta-
galactosidase, viral polypeptides such as herpes gD

10 protein, and the like.

Immunogenic fusions are produced by cross-
linking in vitro or by recombinant cell culture
transformed with DNA encoding an immunogenic
polypeptide. It is preferable that the immunogenic

15 fusion be one in which the immunogenic sequence is

joined to or inserted into AFM or fragment thereof by a
peptide bond(s) . These products therefore consist of a
linear polypeptide chain containing AFM epitope and at
least one epitope foreign to AFM. It will be understood

20 that it is within the scope of this invention to

introduce the epitopes- anywhere within the AFM molecule
or fragment thereof.

Such fusions are conveniently made in
recombinant host cells or by the use of bifunctional

25 cross-linking agents. The use of a cross-linking agent
to fuse AFM to the immunogenic polypeptide is not as
desirable as a linear fusion because the cross-linked
products are not as easily synthesized in structurally
homogeneous form.

30 These immunogenic insertions are particularly

useful when formulated into a pharmacologically
acceptable carrier and administered to a subject in
order to raise antibodies against AFM, which antibodies
in turn are useful in diagnostics or in purification of

35 AFM by immunoaf f inity techniques known per se . In

diagnostic applications, the antibodies will typically

WO 95/27059

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be bound to or associated with a detectable group,
examples of which are well known to those skilled in the
art. Immunoaf f inity techniques could be used, for
example, to purify AFM.
5 Other fusions, which may or may not also be

immunologically active, include fusions of the mature
AFM sequence with a signal sequence heterologous to AFM,
and fusions of AFM to polypeptides having enhanced
plasma half life (ordinarily > about 20 hours) such as

10 immunoglobulin chains or fragments thereof.

Signal sequence fusions are employed in order
to more expeditiously direct the secretion of AFM. The
heterologous signal replaces the native AFM signal, and
when the resulting fusion is recognized, i.e. processed

15 and cleaved by the host cell, AFM is secreted. Signals
are selected based on the intended host cell, and may
include bacterial yeast, mammalian and viral sequences.
The native AFM signal or the herpes gD glycoprotein
signal is suitable for use in mammalian expression

20 systems.

Substantial variants are those in which at
least one residue in the FIG. 2 sequence has been
removed and a different residue inserted in its place.
Such substitutions generally are made in accordance with
25 the following Table 1 when it is desired to finely
modulate the characteristics of AFM.

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TABLE 1

Original Residue Exemplary Substitutions

Ala

Ser

Arg

Lys

Asn

Gin;

His

Asp

Glu

Cys

Ser;

Ala

Gin

Asn

Glu

Asp

Gly

Pro

His

Asn;

Gin

lie

Leu;

Val

Lieu

116 ,

val

Lys

Arg;

Gin;

Met

Leu;

Phe

Met;

Leu;

Ser

Thr

Ser

Trp

Tyr

Trp;

Phe

Val

lie;

Leu

Substantial changes in function or
immunological identity are made by selecting
substitutions that are less conservative than those in
5 Table 1, i.e., selecting residues that differ more
significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobic it y of the
10 molecule at the target site or (c) the bulk of the side
chain. The substitutions which in general are expected
to produce the greatest changes in AFM properties will

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be those in which (a) a hydrophilic residue,, e.g. seryl
or threonyl, is substituted for (or by) a hydrophobic
residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or
alanyl; (b) a cysteine or proline is substituted for (or
5 by) any other residue; (c) a residue having an

electropositive side chain, e.g., lysyl, arginyl, or
histidyl, is substituted for (or by) ah electronegative
residue, e.g., glutamyl or aspartyl; or (d) a residue
having a bulky side chain, e.g., phenylalanine, is

10 substituted for (or by) one not having a side chain,
e.g. glycine.

Some deletions, insertions, and substitutions
will not produce radical changes in the characteristics
of AFM molecule. However, when it is difficult to

15 predict the exact effect of the substitution, deletion,
or insertion in advance of doing so, for example when
modifying the AFM extracellular domain or an immune
epitope, one skilled in the art will appreciate that the
effect will be evaluated by routine screening assays.

20 Another class of AFM variants are deletional

variants. Deletions are characterized by the removal of
one or more amino acid residues from AFM sequence.
Deletions from the AFM C-terminal or the N-terminal,
which preserve the biological activity or immune cross-

25 reactivity of AFM are suitable.

Deletions of cysteine or other labile residues
also may be desirable, for example in increasing the
oxidative stability of AFM. Deletion or substitutions
of potential proteolysis sites, e.g. Arg-Arg, is

30 accomplished by deleting one of the basic residues or
substituting one by glutaminyl or histidyl residues.

Preferably, the variants represent
conservative substitutions. It will be understood that
some variants may exhibit reduced or absent biological

35 activity. These variants nonetheless are useful as

WO 95/27059

PCT/US95/04075

standards in immunoassays for AFM so long as they retain
at least one immune epitope of AFM,

Glycosylation variants are included within the
scope of AFM. They include variants completely lacking
5 in glycosylation (nonglycosylated) and variants having
at least one less glycosylated site than the native form
(deglycosylated) as well as variants in which the •
glycosylation has been changed. Additionally ,
unglycosylated AFM which has the amino acid sequence of

10 the native AFM is produced in recombinant prokaryotic
cell culture because prokaryotes are incapable of
introducing glycosylation into polypeptides .

Glycosylation variants are produced by
selecting appropriate host cells or by in vitro methods.

15 Yeast , for example, introduce glycosylation which varies
significantly from that of mammalian systems.
Similarly, mammalian cells having a different species
(e.g. hamster, murine, insect, porcine, bovine or ovine)
or tissue origin (e.g. lung, lymphoid, mesenchymal or

20 epidermal) than the source of AFM are routinely screened
for the ability to introduce variant glycosylation as
characterized for example by elevated levels of mannose
or variant ratios of mannose, fucose, sialic acid, and
other sugars typically found in mammalian glycoproteins .

25 In vitro processing of AFM typically is accomplished by
enzymatic hydrolysis, e.g. neuraminidase digestion.

AFM isolated from natural sources or produced
recombinantly will generally contain bound lipids. The
nature of the bound lipids is expected to depend on the

30 source of the AFM. Delipidated versions of AFM may be
prepared by standard delipidation methods known in the
art, especially the art relating to ALB where
delipidation is a common procedure. One preferred
method involves extracting an aqueous solution of AFM

35 with a solvent capable of dissolving lipids, such as 1-

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butanol or diisopropyl ether. Example 8 presents a
specific exemplary method for delipidation .

Certain post-translational derivatizations are
the result of the action of recombinant host cells on
5 the expressed polypeptide. Glutaminyl and asparaginyl
residues are frequently post-translationally deamidated
to the corresponding glutamyl and aspartyl residues.
Alternatively, these residues are deamidated under
mildly acidic conditions. Either form of these residues

10 falls within the scope of this invention.

Other post-translational modifications include
hydroxylation of proline and lysine, phosphorylation of
hydroxyl groups of seryl or threonyl residues,
methylation of the a-amino groups of lysine, arginine,

15 and histidine side chains (T ♦ E. Creighton, Proteins:
Structure and Molecular Properties, (W. H. Freeman &
Co.), San Francisco: 79-86 (1983), acetylation of the
N-terminal amine and, in some instances, amidation of
the C-terminal carboxyl.

Oligonucleotides relating to AFM

DNA encoding AFM is synthesized by in vitro
methods or is obtained readily from human liver cDNA
libraries. The means for synthetic creation of the DNA

25 encoding AFM, either by hand or with an automated

apparatus, are generally known to one of ordinary skill
in the art, particularly in light of the teachings
contained herein. As examples of the state of the art
relating to polynucleotide synthesis, one is directed to

30 Maniatis et al., Molecular Cloning-A Laboratory Manual ,
Cold Spring Harbor Laboratory (1984) , and Horvath et
al., An Automated DNA Synthesizer Employing
Deoxynucleoside 3 '-Phosphoramidites, Methods in
Enzymology 154: 313-326 (1987).

35 Alternatively, to obtain DNA encoding AFM from

sources other than murine or human, since the entire DNA

WO 95/27059 ^ PCT/US95/04075

sequence for the preferred embodiment of AFM is given ,
one needs only to conduct hybridization screening with
labelled DNA encoding AFM or fragments thereof (usually,
greater than about 20 , and ordinarily about 50bp) in
5 order to detect clones T which contain homologous

sequences in the cDNA libraries derived from the liver
of the particular animal, followed by analyzing the
clones by restriction enzyme analysis and nucleic acid
sequencing to identify full-length clones. ' If full

10 length clones are not present in the library, then

appropriate fragments are recovered from the various
clones and ligated at restriction sites common to the
fragments to assemble a full-length clone. DNA encoding
AFM from other animal species is obtained by probing

15 libraries from such species with the human sequences, or
by synthesizing the genes in vitro.

Included within the scope hereof are nucleic
acid sequences that hybridize under stringent conditions
to a fragment of the DNA sequence in FIG. 1, which

20 fragment is greater than about 10 bp, preferably 20-50
bp, and even greater than 100 bp. Also included within
the scope hereof are nucleic acid sequences that
hybridize under stringent conditions to a fragment of
AFM. "Stringent" is used to refer to conditions that

25 are commonly understood in the art as stringent. An

exemplary set of conditions include a temperature of 60
- 70°C, (preferably about 65°C) and a salt concentration
of 0.70M to 0.80M (preferably about 0.75M).

Included also within the scope hereof are

30 nucleic acid probes which are capable of hybridizing

under stringent conditions to the cDNA of AFM or to the
genomic gene for AFM (including introns and 5 1 or 3 1
flanking regions extending to the adjacent genes or
about 5,000 bp,, whichever is greater).

WO 95/27059 ^ PCT/US95/04075

Recombinant Expression of AFM

In general , prokaryotes are used for cloning
of DNA sequences in constructing the vectors useful in
the invention. For example, E. coli K12 strain 2 94
5 (ATCC No. 3144 6) is particularly useful. Other

microbial strains which may be used include E. coli B
and E coli X1776 (ATCC No. 31537) . Alternatively,' in
vitro methods of cloning, e.g. polymerase chain
reaction, are suitable.

10 AFMs of this invention are expressed directly

in recombinant cell culture as an N-terrninal methionyl
analog, or as a fusion with a polypeptide heterologous
to AFM, preferably a signal sequence or other
polypeptide having a specific cleavage site at the N-

15 terminus of AFM. For host prokaryotes that do not
process AFM signal, the signal is substituted by a
prokaryotic signal selected for example., from the group
of the alkaline phosphatase, penicillinase, or heat
stable enterotoxin II leaders. For yeast secretion the

20 human AFM signal may be substituted by the yeast

invertase, alpha factor or acid phosphatase leaders. In
mammalian cell expression the native signal is
satisfactory for mammalian AFM, although other mammalian
secretory protein signals are suitable, as are viral

25 secretory leaders, for example the herpes simplex gD
signal .

AFM may be expressed in any host cell-, but
preferably are synthesized in mammalian hosts. However,
host cells from prokaryotes, fungi, yeast, insects and

30 the like are also are used for expression. Exemplary

prokaryotes are the strains suitable for cloning as well
as E. coli W3110 (F-, 1-, prototrophic, ATTC No. 7325),
other enterobacteriaceae such as Serratia marescans,
bacilli and various pseudomonads . Preferably the host

35 cell should secrete minimal amounts of proteolytic
enzymes .

WO 95/27059 ° X PCT/US95/04075

Expression hosts typically are transformed
with DNA encoding AFM which has been ligated into an
expression vector. Such vectors ordinarily carry a
replication site (although this is not necessary where
5 chromosomal integration will occur) . Expression vectors
also include marker sequences which are capable of
providing phenotypic selection in transformed cells, as
will be discussed further below. Expression vectors
also optimally will contain sequences which are useful

10 for the control of transcription and translation, e.g.,
promoters and Shine-Dalgarno sequences (for prokaryotes)
or promoters and enhancers (for mammalian cells) . The
promoters may be, but need not be, inducible..

Promoters suitable for use with prokaryotic

15 hosts illustratively include the B-lactamase and lactose
promoter systems (Chang et al., Nature, 275, 615 (1978);
and Goeddel et al., Mature 281, 544 (1979)), alkaline
phosphatase, the tryptophan (trp) promoter system
(Goeddel, Nucleic Acids Res. 8: 4057 (1980) and EPO

20 Appln. Publ. No. 36,776) and hybrid promoters such as
the tac promoter (H. de Boer et al., Proc. Natl. Acad.
Sci. USA 80, 21-25 (1983)). However, other functional
bacterial promoters are suitable. Their nucleotide
sequences are generally known, thereby enabling 1 a

25 skilled worker operably to ligate them to DNA encoding
AFM (Siebenlist et al., Cell 20, 269 (1980)) using
linkers or adapters to supply any required restriction
sites. Promoters for use in bacterial systems also will
contain a Shine-Dalgarno (S.D.) sequence operably linked

30 to the DNA encoding AFM.

In addition* to prokaryotes, eukaryotic
microbes such as yeast or filamentous fungi are
satisfactory. Saccharomyces cerevisiae is the most
commonly used eukaryotic microorganism, although a

35 number of other strains are commonly available.

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Suitable promoting sequences for use with
yeast hosts include the promoters for 3-phosphoglycerate
kinase (Hitzeman et al., J. Biol. Chem. 255, 2073
(1980)) or other glycolytic enzymes (Hess et al.,
5 J. Adv. Enzyme Reg. 7: 149 (1968); and Holland,
Biochemistry 11, 4900 (1978)), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase phosphof ructokinase, glucose-6-
phosphate isomerase, 3-phosphoglycerate mutase, pyruvate

10 kinase, triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase.

Other yeast promoters, which are inducible
promoters having the additional advantage of .
transcription controlled by growth conditions, are the

15 promoter regions for alcohol dehydrogenase 2,

isocytochrome C, acid phosphatase, degradative enzymes
associated with nitrogen metabolism, metallothionein,
glyceraldehyde 3-phosphate dehydrogenase, and enzymes
responsible for maltose and galactose utilization.

20 Suitable vectors and promoters for use in yeast

expression are further described in R. Hitzeman et al.,
European Patent Publication No. 73,657A.

Expression control sequences are known for
eukaryotes. Virtually all eukaryotic genes have an AT-

25 rich region located approximately 25 to 30 bases

upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the
start of transcription of many genes is a CXCAAT region
where X may be any nucleotide. At the 3 f end of most

30 eukaryotic genes is an AATAAA sequence which may be the
signal for addition of the poly A tail to the 3 1 end of
the coding sequence. All of these sequences are
inserted into mammalian expression vectors.

Suitable promoters for controlling

35 transcription from vectors in mammalian host cells are
readily obtained from various sources, for example, the

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PCT/US95/04075

genomes of viruses such as polyoma virus. SV40,
adenovirus, MMV (steroid inducible), retroviruses (e.g.
the LTR of HIV) , hepatitis-B virus and most preferably
cytomegalovirus, or from heterologous mammalian
5 promoters, e.g. the beta act in promoter. The early and
late promoters of SV40 are conveniently obtained as an
SV40 restriction fragment which also contains the SV40
viral origin of replication. Fiers et al., Nature 273,
113 (1978) . The immediate early promoter of the human
10 cytomegalovirus is conveniently obtained as a Hindlll E
restriction fragment. Greenaway, P. J. et al., Gene 18,
355-360 (1982) .

Transcription of a DNA encoding AFM by higher
eukaryotes is increased by inserting an enhancer
15 sequence into the vector. Enhancers are cis-acting

elements of DNA, usually about from 10-300 bp, that act
on a promoter to increase its transcription. Enhancers
are relatively orientation and position independent
having been found 5 f (Laimins, L. et al., PNAS 78, 993
20 (1981)) and 3' (Lusky, M. L., et al., Afol. Cell Bio. 3,
1108 (1983) to the transcription unit, within an intron
(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as
within the coding sequence itself (Osborne, T. F . ,
et al., Afol. Cell Bio. 4, 1293 (1984)). Many enhancer
25 sequences are now known from mammalian genes (globin,
elastase, albumin, a-f etoprotein and insulin) .
Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40
enhancer on the late side, of the replication origin (bp
30 100-270) , the cytomegalovirus early promoter enhancer,
the polyoma enhancer' on the late side of the replication
origin, and adenovirus enhancers.

Expression vectors used in eukaryotic host
cells (yeast, fungi, insect, plant, animal, human or
35 nucleated cells from other multicellular organisms) will
also contain sequences necessary for the termination of

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PCT/US95/04075

transcription which may affect mRNA expression. These
regions are transcribed as polyadenylated segments in
the untranslated portion of the mRNA encoding AFM. The
3' untranslated regions also include transcription
5 termination sites .

Expression vectors may contain a selection
gene, also termed a selectable marker. Examples of
suitable selectable markers for mammalian cells are
dihydrofolate reductase (DHFR) , thymidine kinase (TK) or
10 neomycin.

Suitable eukaryotic host cells for expressing
AFM include monkey kidney CV1 line transformed by SV40
(COS-7, ATCC CRL 1651) ; human embryonic kidney line (293
or 293 cells subcloned for growth in suspension culture,

15 Graham, F. L. et al., J. Gen Virol, 36: 59 (1977)); baby
hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster
ovary-cells-DHFR (CHO, Urlaub and Chasin, PNAS (USA) 77,
4216, (1980)); mouse Sertoli cells (TM4, Mather, J, P.,
Biol. Reprod. 23, 243-251 (1980)); monkey kidney cells

20 (CV1 ATCC CCL 70); African green monkey kidney cells
(VERO-76 , ATCC CRL-1587) ; human cervical carcinoma
cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,
ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver

25 cells (Hep G2, HB 8065); mouse mammary tumor (MMT

060562, ATCC CCL51) ; and, TRI cells (Mather, J. P. et
al., Annals N.Y. Acad. Sci. 383, 44-68 (1982)).

Construction of suitable vectors containing
the desired coding and control sequences employ standard

30 ligation techniques. Isolated plasmids or DNA fragments
are cleaved, tailored,* and. religated in the form desired
to form the plasmids required.

Host cells are transformed with the expression
vectors of this invention and cultured in conventional

35 nutrient media modified as appropriate for inducing
promoters, selecting transf ormants or amplifying AFM

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gene. The culture conditions, such as temperature, pH
and the like, are those previously used with the host
cell selected for expression, and will be apparent to
the ordinarily skilled artisan.
5 The host cells referred to in this disclosure

encompass cells in in vitro culture as well as cells
which are within a host animal.

"Transformation" means introducing DNA into an
organism so that the DNA is replicable, either as an

10 extrachromosomal element or by chromosomal integration.
The method used herein for transformation of the host
cells may be, for example, the method of Graham, F. and
van der Eb, A., Virology 52, 456-457 (1973). However,
other methods for introducing DNA into cells such as by

15 nuclear injection or by protoplast fusion may also be
used. If prokaryotic cells or cells which contain
substantial cell wall constructions are used, the
preferred method of transfection is calcium treatment
using calcium chloride as described by Cohen, F . N.

20 et al., Proc. Natl. Acad. Sci. (USA) 69, 2110 (1972).

"Transfection" refers to the introduction of
DNA into a host cell whether or not any coding sequences
are ultimately expressed. Numerous methods of
transfection are known to the ordinarily skilled

25 artisan, for example, CaP04 and electroporation .
Transformation of the host cell is indicative of
successful transfection.

Recovery and Purification of AFM

30 AFM is recovered and purified from recombinant

cell cultures by known methods, including ammonium
sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose
chromatography, immunoaf f inity chromatography,

35 hydroxyapatite chromatography, lectin chromatography,
hydrophobic interaction chromatography, and gel

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filtration chromatography. Other known purification
methods within the scope of this invention utilize, for
example, immobilized carbohydrates, epidermal growth
factor, or complement domains. Moreover, reverse-phase
5 HPLC and chromatography using anti-AFM antibodies are
useful for the purification of AFM. AFM may preferably
be purified in the presence of a protease inhibitor such
as PMSF. A specific preferred method of purifying AFM
is found in Example 1.

Uses of AFM

As mentioned above, in view of the homology
between AFM and each of ALB, AFP and VDB, it is expected
that AFM and variants thereof will have identical or

15 similar biological activities and utilities. In general,
members of the albumin family show a high propensity to
bind and transport a wide variety of substances through
the body including fatty acids, hormones, enzymes, dyes,
trace metals and drugs. It is expected that a

20 preparation of AFM which has its endogenous lipids

removed could be used to reduce high concentrations of
free fatty acids (hyperlipidemia) found in disease
states such as acute pancreatitis, ARDS, sepsis and
atherosclerosis .

25 Similar to proposed functions for ALB, AFM can

also potentially be used as an antioxidant. AFM is
expected to act as an antioxidant by sequestering metal
ions and preventing those ions from accelerating- free-
radical reactions (ie. decomposition of lipid peroxides

30 to peroxy and alkoxy radicals; formation of hydroxyl
radical from hydrogen peroxide) or AFM may act to
directly inactivate hydrogen peroxide, hydroxyl radicals
and hypochlorous acid. The detoxification of oxygen
metabolites by AFM can limit the detrimental effects

35 that unbound oxygen metabolites have in inactivating
beneficial anti-proteases (ai-antiproteinase and a x -

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antitrypsin) and in damaging DNA, proteins and lipids.
Thus, AFM may be used to ameliorate ischaemia-
reperfusion injury, rheumatoid arthritis, ARDS,
cardiopulmunary bypass, sepsis and any other diseases or
5 tissue damage caused by an excess production of oxygen
metabolites from leukocytes and/or arachidonic acid
metabolism.

Another use of AFM is based on the finding
that albumin family proteins bind and detoxify a wide

10 variety of toxic products including man-made drugs .
Thus, AFM can ameliorate the effects of toxic plasma
substances released as a result of inflammation and can
be conjugated to toxic pharmaceuticals so as to minimize
detrimental activities of the drug.

15 AFM may also have anticoagulant properties as

has been reported for ALB. AFM may inhibit platelet
aggregation and thus could be a useful additive to
resuscitation fluids in disease states characterized by
increased platelet aggregation such as sepsis,

20 hemmorrhagic shock and burn injury.

Serum AFM levels may increase or decrease due
to a particular pathological condition as has been
observed with AFP. Thus, calculation of serum AFM levels
using antibodies described in Example 11 will be useful

25 in diagnosing specific human diseases.

Administ ration of AFM

For administration in vivo, AFM is placed into

sterile, isotonic formulations, and administered by
30 standard means well known in the field. The formulation

of AFM is preferably liquid, and is ordinarily a

physiologic salt solution containing 0.5-10 mM calcium,

non-phosphate buffer at pH 6.8-7.6, or may be

lyophilized powder.
35 It is envisioned that intravenous delivery, or

delivery through catheter or other surgical tubing will

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be the primary route for therapeutic administration.
Alternative routes include tablets and the like,
commercially available nebulizers for liquid
formulations, and inhalation of lyophilized or
5 aerosolized receptors. Liquid formulations may be

utilized after reconstitution from powder formulations.

AFM may also be administered via microspheres,
liposomes, other microparticulate delivery systems or
sustained release formulations placed in certain tissues

10 including blood.

The dose of AFM administered will be dependent
upon the properties of AFM employed, e.g. its activity
and biological half-life, the concentration of AFM in
the formulation, the administration route for AFM, the

15 site and rate of dosage, the clinical tolerance of the
patient involved, the pathological condition afflicting
the patient and the like, as is well within the skill of
the physician;

AFM may also be administered along with other

20 pharmacologic agents such as antibiotics, anti-
inflammatory agents, and anti-tumor agents. It may also
be useful to administer AFM along with other therapeutic
proteins such as gamma-interf eron and other
immunomodulators .

T.iaands of AFM

It is also posible that the AFM of the present
invention will have an additional natural ligand or
ligands (other than the lipids discussed herein) and

30 that such ligands will be capable of modulating the

biological activities* of AFM in vivo. One of ordinary
skill may be able to utilize the teachings of the
present invention to screen for ligands of the AFMs
disclosed herein and then isolate and purify them, e.g.,

35 by immunochromatography using AFM or a variant of AFM
bound to a solid support.

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Antibodies to AFM

Also included within the scope of the
invention are antibodies (e.g., monoclonal and
5 polyclonal antibodies, single chain antibodies, chimeric
antibodies, CDR-grafted antibodies and the like) or
other binding proteins which are specific Ifor AFM or its
variants. Antibodies can be developed using natural or
recombinant AFM or AFM variants or cells expressing such

10 molecules on their surfaces . Active fragments of such
antibodies are also contemplated.

The antibodies are useful for purifying the
polypeptides described herein, or identifying and
purifying cells producing the polypeptides on their

15 surfaces. The antibodies could also be used to modulate
(e.g., block, inhibit or stimulate) ligand binding to
AFM. Anti-idiotypic antibodies are also contemplated.
Assays for detection and quantitation of AFM on cell
surfaces and in fluids (such as serum, blood,

20 cerebrospinal fluid, urine, semen, milk, and tears) may
involve a single antibody or a "sandwich" assay format.

* * *

25 It is understood that the application of the

teachings of the present invention to a specific problem
or situation will be within the capabilities of one
having ordinary skill in the art in light of the ■
teachings contained herein. Examples of the products of

30 the present invention and representative processes for

their isolation, use, and manufacture appear below. All
literature and patent citations herein are expressly
incorporated by reference.

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EXAMPLES

example 1: Lipopolvsaccharide (LPS) Neutralization Assay

5 A. Formation of ELPS .

Sheep erythrocytes are first coated with S.
minnesota Re595 LPS (List Biological) as previously
described (J. Ex. Med. 170:1231) . The concentration of
LPS used to coat erythrocytes is 10 mg/8 x 10 7
10 erythrocytes. After coating with LPS, erythrocytes are
resuspended in 5 mM veronal buffer, .pH 7.5, containing
150 mM NaCl, 0.1% gelatin and 1 mM EDTA (EDTAGVB 2 ~) to a
concentration of 1x10 s cells/ml.

15 B. Opsonization of ELPS.

ELPS are opsonized using normal human plasma (NHP)
as a source of opsonizing proteins (septin) (J. Ex. Med.
176:719) or LBP (J. Ex. Med. 170:1231). NHP is diluted
1:100 in phosphate buffered saline without divalent

20 cations containing 1 mM EDTA (PDEDTA) and then mixed 1:1
with ELPS (1 x 10 8 cells /ml) . The mixture is then
incubated 10 minutes at 37°C, spun down for 1 minute at
800x g in a swinging bucket centrifuge. Cells are
washed 2x with EDTAGVE 2 " and resuspended to a final

25 concentration of 1 x 10 8 cells/ml in EDTAGVB 2 ~.

Erythrocytes treated as above are denoted E-septins.

C. Assay of Samples for LPS Neutralizing Activity.

The following step measures the ability of a given
30 sample to reduce the amount of binding of LPS coated

erythrocytes to macrophages in a CD-14 dependent manner.

Dilutions of samples to be tested are made in
PDEDTA. E-septins are incubated 1:1 with sample
dilutions at 37°C for 4 0 minutes. One sample is
35 included in which E-septin is incubated with PDEDTA

alone as a control. Following incubation, E-septins are

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spun down in a swinging bucket centrifuge at 800x g for
1 minute and resuspended in EDTAGVB 2 " to a concentration
of 1 x 10 8 cells/ml. E-septins treated in this manner
are denoted E-septin 1 . Five ml of the resuspended E-
5 sept ins 1 are added to a macrophage monolayer (see below)
in a 60 well terasaki plate (NUNC, Inc.) . The plate is
incubated 20 minutes at room temperature followed by
inversion and incubation another 12 minutes at room
temperature to separate unbound E-septins 1 from the

10 macrophages. The plate is then dipped in a beaker of
cold PBS with no divalent cations containing 0.001%
azide to wash off unbound erythrocytes. Binding of E-
septins 1 is evaluated by phase contrast microscopy.
Binding is expressed as the attachment index which

15 denotes the number of erythrocytes bound per 100

macrophages. LPS neutralizing activity of the samples
is evaluated by comparing the binding of E-septins 1
incubated with dilutions of the sample in PDEDTA to E-
septin 1 incubated with PDEDTA alone. Activity is

20 measured in units/ml. This represents the dilution at

which the sample reduces E-septin 1 binding by 50%- in the
standard assay.

D. Macrophage Monolayer.

25 Monocyte derived macrophages are obtained by

culturing human monocytes in teflon beakers as described
(J. Ex. Med. 156:1149). On the day of the assay,
macrophages are removed from the beaker, washed with PBS
containing divalent cations (PBS 2 + ) and resuspended in

30 PBS 2 + containing 0.5 mg/ml human serum albumin (Armour
Pharm.), 0.3 U/ml aprotinin (Sigma) and 3 mM glucose
(HAP buffer) to 1 x 10 6 cells/ml. Thirty minutes prior
to addition of E-septins 1 to the terasaki plate,
macrophage monolayers are formed by adding 5 ml of cells

35 and 5 ml of HAP buffer to each well. Immediately prior

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to addition of E-septins 1 , the plate is flooded with
PDEDTA and the wells are lightly aspirated.

F.YAMPT.K ?: Purification of AFM
5 All steps , except where noted were done at

controlled room temperature. All buffers were made with
pyrogen free water and sterile filtered prior to use.
One unit of fresh frozen plasma (FFP) was thawed by
immersion of the packet in a room-temperature water

10 bath. The thawed plasma was first transferred to a

graduated cylinder, the volume noted, and then poured
into a beaker. The FFP was then stirred magnetically
while the beaker was immersed in a ice-water bath. To
the FFP/ sufficient 3 . 9M ammonium sulfate (4°C) was

15 slowly added to achieve a final ammonium sulfate

concentration of 1.6M. This mixture was allowed to stir
at 4°C for an additional 4 hours. The mixture was then
centrifuged at 10,000 x g for 60 minutes. The
supernatant was recovered and allowed to come to room

20 temperature by immersion in a room temperature water
bath.

A portion (250 ml) of the ammonium sulfate
supernatant was loaded onto a 2.6 x 10cm Phenyl
Sepharose HP column previously equilibrated with' 50mM

25 sodium phosphate, 1.6M ammonium sulfate; pH 7.5.

Loading, wash and elution steps were done at a linear
flow rate of 40cm/hr. Following sample loading the
column was washed in turn with 50mM sodium phosphate,
1.6M ammonium sulfate; pH 7.5 and 50mM sodium phosphate;

30 pH 7.5. Elution of activity (see Example 1) was

accomplished by the introduction of water. The pH of
the water eluate was adjusted to 8.2 with 0.1M Tris base
and loaded, at a flow rate of 150cm/hr, onto a 2.6 x
10cm Q Sepharose HP column previously equilibrated with

35 20mM Tris-HCl; pH 8.2. Following sample loading the
column was thoroughly washed with first 20mM Tris-HCl;

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pH 8.2 and then 0.1M sodium chloride, 20mM Tris-HCl; pH
8.2. The column was then resolved with a 0.33mM/ml
linear gradient of sodium chloride ranging from 0 . 1 to
0.3M, followed by a 7mM/ml linear gradient of sodium
5 chloride ranging from 0.3M to 1.0M in 20mM Tris-HCl; pH
8.2. Active fractions were pooled (30 ml).

One third of the pooled activity from above
was loaded onto a Superdex 200 prep column (2.6 x 60cm)
previously equilibrated with phosphate buffered saline;

10 pH 7.4. The column was loaded and resolved at a flow
rate of lml/min. The fractions were analyzed for
activity and by reducing SDS-PAGE. A pool (8mi) of the
appropriate fractions showed two major protein bands by
reducing SDS PAGE at a molecular weight of 87 kd and 28

15 kd. Two milliliters of the above pool was submitted for
sequence analysis. The 28 kd protein had the same N-
terminus as ApoAl and the 87 kd protein had a novel N-
terminus (see below) . The remaining 6ml of the Superdex
200 prep pool was loaded onto a Superdex 200 prep

20 column (2. 6 x 60cm) previously equilibrated with 1%

sodium deoxycholate, 0.15M sodium chloride, 50mM Tris-
HCl; pH 8.5. The column was loaded and resolved at a
flow rate of lml/min. The fractions were analyzed by
SDS-PAGE. Those fractions corresponding to either the

25 87 kd or the 28 kd proteins were separately pooled (16ml
each) .

Four milliliters of each pool was diafiltered
against 50mM Tris-HCl; pH 8.5 to remove excess sodium
deoxycholate and concentrated to approximately O.lmg/ml.

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EXAMPLE 3: Amino Acid Sequence Analysis of N-terminus

and Proteolytic Fragments of Purified AFM

To obtain the N-terminal sequence , 10 ug of
purified AFM was loaded on the reverse phase portion of
5 a precycled biphasic sequencing column. The column was
loaded on a Hewlett Packard G1004A protein sequencer
with on-line phenylthiohydantoin amino acid analysis
performed with a Hewlett Packard 1090 high performance
liquid chromatography (HPLC) . The N-terminal amino acid
10 sequence obtained from the protein was :

LPTQPRDIENFXSTQKFIEDNIEYITIIAFAQYVQ (SEQ ID NO: 6) ,

where "X" represents an unassignable amino acid.

15 To obtain sequences from AFM tryptic peptide

fragments, 100 mg of AFM was dissolved in 8M urea with
0.4 M ammonium bicarbonate, then reduced with DTT and
carboxymethylated with iodoacetic acid. The sample was
subsequently diluted with water to adjust the urea

20 concentration to 2M, then digested with sequencing grade
trypsin (Boehringer Mannheim) at 37°C for 18 h with an
enzyme to substrate ratio of 1:50. The digested protein
was injected on a Hewlett Packard 1090 HPLC equipped
with a 4.6 x 250 mm C18 reverse phase column (Vydac) .

25 Elution was performed using a linear gradient of

acetonitrile with 0.1 % trif luoroacetic acid at a flow
rate of 0.75 ml/min. Elution was monitored at 214 nm and
fractions were collected. Selected fractions were run on
the Hewlett Packard G1004A protein sequencer as

30 described above. The amino acid sequences obtained from
select tryptic fragments are summarized in Table 1.

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Table 1: Trypgip fragments

FX18 YHYLIR (SEQ ID NO: 7)

5 FX20 FTFEYSR (SEQ ID NO: 8)

FX27 F TD SENVCQERD ADP (SEQ ID NO: 9)

FX29 IVQIYKDLLR (SEQ ID NO: 10)

FX32 IAPQLSTEELVSLGE (SEQ ID NO: 11)

FX37 RHPDLSIPELLR (SEQ ID NO: 12)

15 FX45 ESLLNHFLYEVAR (SEQ ID NO: 13)

FX53 RNP F VF AP TLLT VAVHFEE VAKS C C (SEQ ID NO: 14)

20 EXAMPLE 4: Isolation of AFM cDNA

The polymerase chain reaction (PCR) was used
to amplify a portion of the AFM gene from which an exact
probe could be derived. A PCR (PCR 1) was first
performed with fully degenerate primers specifying the

25 sense strand for the N-terminal amino acid sequence

QKFIEDN (SEQ ID NO: 15) [5 f ACG CTG AAT TCG CCA (GA)AA
(GA)TT (CT)AT (ATC)GA (GA)GA (CT)AA] (SEQ ID NO:' 16) and
the antisense strand for a portion of the FX 29 tryptic
peptide sequence IVQIYKD (SEQ ID NO: 17) [5 1 ACG CTA

30 AGC TTG C(GA)T C(CT)T T (GA) T A (GAT) A T (CT) T G (AGCT) A

C (GAT) A T] (SEQ ID NO:" 18) . One ng of Quick Clone Human
liver cDNA (Clontech, cat. no. 7113-1) was used as the
template in a 100 ml PCR performed in a standard buffer
(Perkin-Elmer Cetus) with 1 mM of each degenerate

35 primer. The cycling parameters used in the PCR were as
follows: 95°C , 8 min (1 cycle); 94°C, 1 min, 34°C, 10

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min, 72°C, 2 min (3 cycles); 94°C, 1 min f 50°C, 1 min,
72°C, 2 min (45 cycles); 72°C, 5 min (1 cycle).

Agarose gel analysis of PCR 1 did not reveal
the amplification of any specific products, thus we
5 utilized an aliquot of PCR 1 as a template for a second
PCR (PCR 2) using a nested primer pair. For this
. experiment, we used fully degenerate primers specifying
the sense strand for the N-terminal amino acid sequence
DNIEYIT (SEQ ID NO: 19) [5* ACG CTG AAT TCG CGA (CT)AA

10 (CT)AT (ATC)GA (GA)TA (CT)AT (ATC)AC] (SEQ ID NO: 20)
and the antisense strand for the FX 20 tryptic peptide
sequence FTFEYS (SEQ ID NO: 21) [5 f ACG CTA AGC TTG
C(GATC)G A (GA) T A(CT)T C(GA)A A (ACGT) G T (GA) A A] (SEQ ID
NO: 22) . PCR 2 was then performed using the same

15 reaction mix and cycling parameters as PCR 1, except for
the substitution of 1 ml of PCR 1 in place of the human
liver cDNA. Analysis of PCR 2 by agarose gel
electrophoresis revealed the amplification of a 1 kb
product .

20 To prepare PCR 2 for DNA sequencing, the

inventors utilized the EcoR I and Hind III sites that
were incorporated into the degenerate primers. These
sites were used to clone the fragment into mpl9
(Boehringer Mannheim) . The 1 kb PCR 2 product cloned in

25 mp!9 (mpl9 AFM) was then sequenced in its entirety from
both strands. Nucleotide sequence analysis of the
fragment confirmed that we had amplified a segment of
AFM cDNA as the sequence was found to have an open
reading frame which encoded 3 tryptic peptides that were

30 already sequenced (Table 1, Fxs 27, 45, 53) . The

nucleotide sequence of the 1 kb fragment was compared to
all sequences in the Genbank database and found to be
unique. We also observed that the partial AFM cDNA had
significant homology with cDNAs reported for ALB family

35 proteins (ALB, AFP and VDB) . Therefore, in order to to
isolate a full-length cDNA encoding AFM and to minimize

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the probability of hybridizing other ALB family genes ,
we screened a human liver cDNA library with an exact
18mer oligonucleotide (5 1 TAT GTG CTA TGG AGG GGC) (SEQ
ID NO: 23) derived from a segment of ATM sequence that
5 was not highly homologous to ALB, AFP and VDB . The
oligonucleotide was end-labeled with 32 Pi and used to
screen a human liver cDNA library (Clontech, cat. no.
HLlllSa) . Positive clones were purified and rescreened
with the same oligonucleotide probe. A single positive

10 clone (17AFM) with a 2.3 kb insert was chosen for

further study and phage DNA was prepared. The 2.3 kb
insert was then sequenced in its entirety from both
strands and verified to encode AFM.

This approach enabled the inventors to isolate

15 a lambda phage (17AFM) containing full-length AFM cDNA.

EXAMPLE 5 : Characterization of AFM

The insert in 17AFM is 2287 bp (FIG. 1)
consisting of a 317 bp 5' untranslated region, a 1797 bp

20 sequence encoding a protein of 599 amino acids and a 173
bp 3 1 untranslated region. The predicted amino acid
sequence of AFM was found to contain all the tryptic
peptides (See Table 1) that had been previously
sequenced from the purified protein. Translation of the

25 AFM cDNA sequence reveals that a 21 amino acid

hydrophobic leader peptide precedes the experimentally
determined N-terminus of mature AFM. The mature AFM is
predicted to have 578 amino acids with a calculated Mr
of 66576 and pi of 5.65. The difference between the

30 calculated Mr of AFM and its apparent molecular weight
on SDS-PAGE is likely -due to glycosylation . AFM has 4
potential sites for N-glycosylation (FIG. 1) and N-
glycanase treatment reduced the apparent Mr of AFM to
65000 when analyzed by reducing SDS-PAGE (data not

35 shown) .

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A comparison of the deduced AFM. amino acid
sequence and other ALB family sequences is shown in FIG.
2 . It is evident there is strong similarity between AFM
and other ALB family members throughout the entire
5 protein. When conserved amino acids are taken into

account , the of AFM to AFP, ALB and VDB is 60.4%, 54.8%
and 41.2% respectively. The distribution of Cys residues
is conserved among ALB family members . The positions of
AFM Cys residues are clearly consistent with this

10 arrangement (FIG. 3) . The Cys residues in ALB family
proteins have also been proposed to form a pattern of
disulfide bridges enabling these proteins to be depicted
as a series of 9 double loops defining 3 structural
domains. FIG. 4 shows that the 34 Cys residues in AFM

15 can be arranged into a pattern of 17 disulf ide-linked

pairs that parallels the domain organization observed in
other ALB family proteins.

EXAMPLE 6: Chromosomal Mapping

20 PCR was performed on a panel of somatic cell

hybrids (Bios Laboratories, cat. no. CP2-02) to identify
the chromosomal location of the gene. For PCR, we
utilized primers (5' CAA CCC TGC TGT GGA CCA C; 5' GCA
CAT ATG TTT TAT CAG CTT T) (SEQ ID NO: 24 and SEQ ID NO:

25 25) that would be expected to amplify an 88 bp fragment
between nt 17 90 and 1878 in the AFM cDNA. Each PCR on
somatic cell hybrid DNA was performed in a standard 100
ml reaction mixture (Perkin-Elmer Cetus) containing 250
ng DNA and a final concentration of 0.1 mM of each

30 primer. The cycling parameters were as follows: 95°C, 5
min (1 cycle); 94°C, 1 min, 56°C, 1 min, 72°C, 1 min (25
cycles); 72°C, 5 min (1 cycle).

Utilizing PCR on a panel of commercially
available somatic cell hybrids (data not shown) , we

35 detected an amplified product in only 2 hybrids. Both of
these hybrids had DNA in common from human chromosomes

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4, 5 and 8. Since PCRs performed on hybrids containing
DNA from chromosomes 5 and 8 did not yield amplified
products, we conclude that AFM resides on human
chromosome 4 along with other ALB family genes.

example 7; stable Expression of rAFM

Two separate PCR f s were performed on AFM- cDNA
to generate 2 overlapping fragments that span the entire
AFM cDNA sequence. The oligonucleotide pair (5' TCA CCT

10 CTA GAC CAC CAT GAA ACT ACT AAA ACT TAC AG + 5 1 AAT TTC
TCA GGA GAT CTT TGT ATA) (SEQ ID NO: 26 and SEQ ID NO:
27) used in the first PCR introduced an Xba I site and a
perfect Kozak sequence preceeding the AFM initiator
codon. The amplified product was subsequently cloned

15 into pGEMT (Promega) to create pD Jll . The

oligonucleotide pair (5 f AAA TAT ACA AAG ATC TCC TGA GAA
+ 5' TCC CGG TCG ACT CAG TTG CCA ATT TTT GGA C) (SEQ ID
NO: 28 and SEQ ID NO: 29) used in the second PCR
introduced a Sal I site following the natural stop codon

20 in AFM and the product was cloned into pGEMT to create
pDJ13. The 3 f end of AFM was then joined to the 5' end
by ligating a Bgl II-Sal I fragment from pDJ13 into
pDJll that had been digested with Bgl II and Sal I. The
resultant plasmid (pDJ14) was then digested with Xba I

25 and Sal I and the entire AFM cDNA was cloned into the
mammalian expression vector pDSRa (European Patent
Application A20398753) that was modified to include
unique Xba I and Sal I sites. The AFM expression vector
was used to transfect a Chinese Hamster Ovary (CHO) cell

30 line deficient in dihydrof olate reductase (CHO D~) .
Transf ectants were selected in medium lacking
hypoxanthine and thymidine. An RNase protection assay
was used to screen for transf ectants that had AFM-
specific mRNA. A single clone was grown without serum as

35 described (See, Bourdrel, L. et al., Protein Expression

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Purif. 4: 130 (1993) to generate conditioned medium (CM)
containing rAFM.

A single stable trans fectant expressing high
levels of AFM-specific mRNA was isolated. Immunoblots
5 performed with AM339 (see Example 12) demonstrates that
this antibody recognizes both rAFM produced from the
transf ectant as well as natural AFM isolated from human
plasma (FIG. 5) . rAFM was purified from CM derived from
this transf ectant (FIG. 6) and SDS-PAGE demonstrated
10 rAFM to be greater than 95% pure with the same N-

terminus as AFM purified from plasma (data not shown) .

EXAMPLE 8: Protein Purification

Serum-free CM was concentrated 10-fold and

15 diafiltered against 25 mM sodium phosphate, pH 7.5 using
a Filtron ultrafiltration apparatus loaded with 10 K
molecular weight cut-off filters. The CM was then
adjusted with 3 . 9 M ammonium sulfate to achieve a final
ammonium sulfate concentration of 1.6 M. This mixture

20 was stirred for 0.5 hr; no precipitate was observed. The
solution was then filtered in succession through 0.45mm
and 0.22mm filters. The resultant filtrate was loaded
onto a Phenyl Sepharose HP column previously
equilibrated with 50 mM sodium phosphate, 1.6 M" ammonium

25 sulfate; pH 7.5. Loading, washing and elution were done
at a linear flow rate of 40 cm/hr . Following sample
loading, the column was washed successively with 50 mM
sodium phosphate, 1.6 M ammonium sulfate, pH 7.5- and 50
mM sodium phosphate, pH 7.5. rAFM was eluted with water

30 and detected by immunoblotting (see below) .

The pH of the water eluate was adjusted to 8.0
with 0.1 M Tris-base and loaded at a flow rate of 100
cm/hr onto a Q Sepharose HP column previously
equilibrated with 20 mM Tris-HCl, pH 8.0. Following

35 sample loading, the column was thoroughly washed with 20
mM Tris-HCl, pH 8.2, then with 0.1 M NaCl, 20 mM Tris-

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HC1, pH 8.2. The column was then resolved with a 3
column volume (CV) linear gradient of NaCl ranging from
0 - 0.3 M, followed by a 1 CV linear gradient of NaCl
ranging from 0.3 M to 1.0 M in 20 mM Tris-HCl, pH 8.0.
5 Fractions containing rAFM were pooled and loaded onto a
Superdex 200 column previously equilibrated with
phosphate-buffered saline pH 7.4. The column was loaded
and resolved at a flow rate of 1 ml/min .

10 EXAMPLE 9: Delioidation of AFM

To one volume of protein solution was added
2.5 volumes of 1-butano/diisopropyl ether (40:60). The
mixture was shaken gently for 30 minutes at room
temperature and then centrifuged for 5 minutes at 500 x

15 g. The aqueous layer was recovered and to this was
added a second 2.5 volumes of 1-butanol/diisopropyl
ether (40:60) . The sample was treated as before and the
aqueous phase recovered. To the twice extracted aqueous
phase was added one volume of diisopropyl ether, the

20 mixture shaken and immediately centrifuged at 250 x g

for 5 minutes . The aqueous layer was recovered and used
as essentially delipidated.

EXAMPLE 10: Immunoblottincr

25 All samples for immunoblotting were

electrophoresed on 4-20% SDS polyacylamide gels (Novex)
and the proteins were then electrophoretically •
transferred to nitrocelluose membranes (Schleicher and
Schuell, cat. no. BA83) . The filters were first treated

30 with polyclonal antibody AM339 that was raised in

rabbits against a synthetic peptide (DLSLREGKFTDSENVC)
(SEQ ID NO: 30) derived from amino acids 304 - 319 in
AFM and then with donkey anti-rabbit Ig linked to
horseradish peroxidase. Immune complexes were visualized

35 by enhanced chemiluminescence according to the
manufacturer's (Amersham) specifications.

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EXAMPLE 1 1; Nucleotide sequence comparisons

Nucleotide sequences were compared to the

Genbank (Release 78.0) sequences using the method of
Pearson and Lipman (Proc. Natl. Acad. Sci. USA 84, 2444-
5 2448 (1988)) in the FAST A program of the Genetics

Computer Group (GCG) , Inc. (Madison, WI) . Comparisons
are shown in FIG. 2A.

EXAMPLE 12: Generation of pep tide antibodies specific
10 f or

Since AFM was similar to at least three other
members of the albumin family, it was possible that
antibodies generated against the entire protein would
also cross react with other albumin family proteins . To

15 create AFM-specific antibodies, we first identified
specific peptides of AFM which were dissimilar to
peptide sequences found in the other albumin family
proteins . These peptide sequences were then synthesized
and the synthetic peptides were used to inject rabbits

20 to obtain polyclonal antisera against each peptide. The
AFM peptides that were synthesized are as follows :

HI EKLVKDMVEYKDRC (SEQ ID NO: 31)

25 The corresponding antibody is referred to as AM384.

aa 43-56 in AFM

CIINSNKDDRPKDLSLR

(SEQ ID NO: 32)

aa 292-308 in AFM

The

corresponding antibody is referred to as AM 609.

DL S LREGKF TD SENVC •

(SEQ ID NO: 30)

aa 304-319 in AFM

The

corresponding antibody is referred to as AM 339.

WO 95/27059 ^ ° PCTAJS95/04075

H4 CQERDADPDTFFAKFT (SEQ ID NO: 33)

aa 319-334 in AFM
The corresponding antibody is referred to as AM 1104.

5 EXAMPLE 1 3: Generation of polyclonal antisera to AFM

Rabbit polyclonal antiserum was generated from
purified AFM by methods that are standard in the art.
The antiserum was found to bind specifically to AFM.

10 Discussion of Examples

ALB family proteins are comprised of three
homologous folding domains and are predicted to have
evolved from an ancestral gene that coded for an
approximately 190 amino acid single domain protein

15 containing 3 double loops formed by 6 disulfide bridges.
The genes in this family have all been mapped to the
4qll-q22 region of chromosome 4. AFM shares significant
homology with ALB family proteins and has Cys residues
consistent with a similar overall 3-domain organization.

20 In addition, AFM has been localized to chromosome 4.
Thus, there is compelling evidence that AFM is the
fourth member of the ALB family.

There are some noteworthy distinctions among
ALB family members. Concentrations in adult serum are 50

25 ng/ml for AFP, 350 mg/ml for VDB, 40 mg/ml for ALB and
30 mg/ml for AFM (data obtained by immunoblot analysis) .
ALB is not glycosylated, AFP and VDB each have 1
potential N-gly cosy lat ion site while AFM has 4 potential
sites. ALB expresses one free thiol group that has been

30 implicated in complex formation with Cys, glutathione,
mercurial and gold compounds. In contrast, AFP and VDB
have an even number of Cys residues and are thought not
to have a free thiol. AFM has an even number of Cys
residues, suggesting that it may not have a free thiol

35 and may not bind glutathione and mercurials as does ALB.

WO 95/27059 ^ PCT/US95/04075

There also are differences in the intradomain
disulfide bonding pattern among ALB family members. VDB
is predicted to have a disulfide bridge in double loop
1A. This bridge is absent in ALB, AFP and AFM. A
5 disulfide bridge domain 2C is common to ALB, VDB and AFM
but is not present in AFP. Thus, while the 4 ALB family
proteins are evolutionarily related, there are clear
differences in the molecular organization of these
proteins .

10 Structural similarities between AFM and other

ALB family members suggest that AFM could scavenge or
transport a variety of ligands. We examined whether
known ligand-binding sites in the sequence of ALB family
proteins were also present in AFM. VDB has a binding

15 site for sterols between amino acids 35-49 and a binding
site for actin between amino acids 373-403. Using the
GCG GAP program and the alignment of FIG. 2A, AFM has
60% similarity and 40% identity between VDB amino acids
35-4 9 but only 32% similarity and 10% identity between

20 VDB amino acids 373-403. Thus, it is possible that AFM
has sterol binding sites (e.g., the amino acids in AFM
that correspond to amino acids 35 - 49 of VDB) but it is
not likely to bind actin. The X-ray crystal structure
of ALB was used to show that ALB binds a variety of

25 ligands (aspirin, warfarin, IS, DIS, TIB, bilirubin)

between amino acids 186-260 in domain 2 A and an array of
ligands (aspirin, diazepam, digit oxin, clofibrate,
ibuprofen, IS, DIS, TIB, long chain fatty acids) between
amino acids 37 9-455 in domain 3A. A GCG GAP comparison

30 between analogous regions in AFM reveals that AFM has
54% similarity and 35% identity in domain 2A and 45%
similarity and 25% identity in domain 3A. These
moderate degrees of similarity make it possible but not
conclusive as to whether AFM binds the same ligands as

35 ALB in domains 2 A and 3A.

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AfrfrrevietiQPS

The abbreviations used in the above Examples
5 are: AFM, afamin; AFP, a-f etoprotein; ALB, human serum
albumin; CHO, Chinese Hamster Ovary; CM, conditioned
medium; CV f column volume; DIS, 3, 5- diiodosalicyclic
acid; DTT, dithiothreitol; HPLC, high performance liquid
chromotography; IS, 5-iodosalicyclic acid; PAGE,
10 polyacrylamide gel electrophoresis; PBS, phosphate-
buffered saline; PCR, polymerase chain reaction; r,
recombinant; TIB, 2,3,5- triioidobenzoic acid; VDP,
vitamin D-binding protein.

The invention now being fully described, it
will be apparent to one of ordinary skill in the art
that many changes and modifications can be made thereto,
20 without departing from the spirit and scope of the
invention as set forth herein.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: AMGEN INC.

(ii) TITLE OF INVENTION: Afamin: A Human Serum Albumin-Like *
Protein

(iii) NUMBER OF SEQUENCES: 33

(iv) CORRESPONDENCE ADDRESS :

(A) ADDRESSEE: Amgen Center, Patent Operations/RRC

(B) STREET: 1840 DeHavilland Drive

(D) STATE: California

(E) COUNTRY: U.S.

(F) ZIP: 91320-1789

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2287 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

<ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME /KEY: CDS

(B) LOCATION: 318.. 2117

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 381. .2114

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 318.. 380

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<xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CCCCGAGTCT CTGCGCCTTC ACATAGTTGT CACAGGACTA AAGCAAATTG ATCCAGGGGG 60

AAACACTGTA GACCGTGTAT ATAAAAACAC TCTATAAACT GCAATGCTCA ATTCTTAGTA 120

TAACTATTGT TGTTGTATTG ATATTTATTA GTATTGGTGC TCACAAAAAG AGTCTAAATT 180

CCATAAGTCT TTATATTCAG GCTACTCTTT ATTTTTGAAA ACTCATTTTC TATCACCTTT 240

TTCTATTTTA CTCCATATTG AGGCCTCATA AATCCAATTT TTTATTTCTT TCTTTTGTAA 300

ATGTGGTTTC TACAAAG ATG AAA CTA CTA AAA CTT ACA GGT TTT ATT TTT 350

Met Lys Leu Leu Lys Leu Thr Gly Phe lie Phe
-21 -20 -15

TTC TTG TTT TTT TTG ACT GAA TCC CTA ACC CTG CCC ACA CAA CCT CGG 398
Phe Leu Phe Phe Leu Thr Glu Ser Leu Thr Leu Pro Thr Gin Pro Arg
-10 -5 15

GAT ATA GAG AAC TTC AAT AGT ACT CAA AAA TTT ATA GAA GAT AAT ATT 446
Asp lie Glu Asn Phe Asn Ser Thr Gin Lys Phe lie Glu Asp Asn lie
10 15 20

GAA TAC ATC ACC ATC ATT GCA TTT GCT CAG TAT GTT CAG GAA GCA ACC 4 94

Glu Tyr lie Thr lie lie Ala Phe Ala Gin Tyr Val Gin Glu Ala Thr
25 30 35

TTT GAA GAA ATG GAA AAG CTG GTG AAA GAC ATG GTA GAA TAC AAA GAC 542 .

Phe Glu Glu Met Glu Lys Leu Val Lys Asp Met Val Glu Tyr Lys Asp
40 - 45 50

AGA TGT ATG GCT GAC AAG ACG CTC CCA GAG TGT TCA AAA TTA CCT AAT 590
Arg Cys Met Ala Asp Lys Thr Leu Pro Glu Cys Ser Lys Leu Pro Asn
55 60 65 70

AAT GTT TTA CAG GAA AAA ATA TGT GCT ATG GAG GGG CTG CCA CAA AAG 638
Asn Val Leu Gin Glu Lys He Cys Ala Met Glu Gly Leu Pro Gin Lys
75 80 85

CAT AAT TTC TCA CAC TGC TGC AGT AAG GTT GAT GCT CAA AGA AGA CTC 686
His Asn Phe Ser His Cys Cys Ser Lys Val Asp Ala Gin Arg Arg Leu
90 95 100

TGT TTC TTC TAT AAC AAG AAA TCT GAT GTG GGA TTT CTG CCT CCT TTC 734
Cys Phe Phe Tyr Asn Lys Lys Ser Asp Val Gly Phe Leu Pro Pro Phe
105 ~ 110 115

CCT ACC CTG GAT CCC GAA GAG AAA TGC CAG GCT TAT GAA AGT AAC AGA 7 82

Pro Thr Leu Asp Pro Glu Glu Lys Cys Gin Ala Tyr Glu Ser Asn Arg
120 ' 125 130

GAA TCC CTT TTA AAT CAC TTT TTA TAT GAA GTT GCC AGA AGG AAC CCA 830
Glu Ser Leu Leu Asn His Phe Leu Tyr Glu Val Ala Arg Arg Asn Pro
135 140 145 150

TTT GTC TTC GCC CCT ACA CTT CTA ACT' GTT GCT GTT CAT TTT GAG GAG 878

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Phe Val Phe Ala Pro Thr Leu Leu Thr Val Ala Val His Phe Glu Glu
155 160 165

GTG GCC AAA TCA TGT TGT GAA GAA CAA AAC AAA GTC AAC TGC CTT CAA 926
Val Ala Lys Ser Cys Cys Glu Glu Gin Asn Lys Val Asn Cys Leu Gin
170 175 180

ACA AGG GCA ATA CCT GTC ACA CAA TAT TTA AAA GCA TTT TCT TCT TAT 974
Thr Arg Ala lie Pro Val Thr Gin Tyr Leu Lys Ala Phe Ser Ser Tyr
185 190 195

CAA AAA CAT GTC TGT GGG GCA CTT TTG AAA TTT GGA ACC AAA GTT GTA 1022
Gin Lys His Val Cys Gly Ala Leu Leu Lys Phe Gly Thr Lys Val Val
200 205 210

CAC TTT ATA TAT ATT GCG ATA CTC AGT CAA AAA TTC CCC AAG ATT GAA 1070
His Phe He Tyr He Ala He Leu Ser Gin Lys Phe Pro Lys He Glu
215 220 225 230

TTT AAG GAG CTT ATT TCT CTT GTA GAA GAT GTT TCT TCC AAC TAT GAT 1118
Phe Lys Glu Leu He Ser Leu Val Glu Asp Val Ser Ser Asn Tyr Asp
235 240 245

GGA TGC TGT GAA GGG GAT GTT GTG CAG TGC ATC CGT GAC ACG AGC AAG 1166
Gly Cys Cys Glu Gly Asp Val Val Gin Cys He Arg Asp Thr Ser Lys
250 255 260

GTT ATG AAC CAT ATT TGT TCA AAA CAA GAT TCT ATC TCC AGC AAA ATC 1214
Val Met Asn His He Cys Ser Lys Gin Asp Ser He Ser Ser Lys He
265 270 275

AAA GAG TGC TGT GAA AAG AAA ATA CCA GAG CGC GGC CAG TGC ATA ATT 1262
Lys Glu Cys Cys Glu Lys Lys He Pro Glu Arg Gly Gin Cys He lie
280 285 290

AAC TCA AAC AAA GAT GAT AGA CCA AAG GAT TTA TCT CTA AGA GAA GGA 1310
Asn Ser Asn Lys Asp Asp Arg Pro Lys Asp Leu Ser Leu Arg Glu Gly
295 300 305 " 310

AAA TTT ACT GAC AGT GAA AAT GTG TGT CAA GAA CGA GAT GCT GAC CCA 1358
Lys Phe Thr Asp Ser Glu Asn Val Cys Gin Glu Arg Asp Ala Asp Pro
315 320 325

GAC ACC TTC TTT GCG AAG TTT ACT TTT GAA TAC TCA AGG AGA CAT CCA 1406
Asp Thr Phe Phe Ala Lys Phe Thr Phe Glu Tyr Ser Arg Arg His Pro.
330 335 340

GAC CTG TCT ATA CCA GAG CTT TTA AGA ATT GTT CAA ATA TAC AAA GAT 1454
Asp Leu Ser He Pro Glu Leu Leu Arg He Val Gin He Tyr Lys Asp
345 350 • " 355

CTC CTG AGA AAT TGC TGC AAC ACA GAA AAC CCT CCA GGT TGT TAC CGT 1502
Leu Leu Arg Asn Cys Cys Asn Thr Glu Asn Pro Pro Gly Cys Tyr Arg
360 365 370

TAC GCG GAA GAC AAA TTC AAT GAG ACA ACT GAG AAA AGC CTC AAG ATG 1550
Tyr Ala Glu Asp Lys Phe Asn Glu Thr Thr Glu Lys Ser Leu Lys Met
375 380 385 390

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GTA CAA CAA GAA TGT AAA CAT TTC CAG AAT TTG GGG AAG GAT GGT TTG 1598
Val Gin Gin Glu Cys Lys His Phe Gin Asn Leu Gly Lys Asp Gly Leu
395 400 405

AAA TAC CAT TAC CTC ATC AGG CTC ACG AAG ATA GCT CCC CAA CTC TCC 1646
Lys Tyr His Tyr Leu lie Arg Leu Thr Lys lie Ala Pro Gin Leu Ser
410 415 420

ACT GAA GAA CTG GTG TCT CTT GGC GAG AAA ATG GTG ACA GCT TTC ACT 1694
Thr Glu Glu Leu Val Ser Leu Gly Glu Lys Met Val Thr Ala Phe Thr
425 430 435

ACT TGC TGT ACG CTA AGT GAA GAG TTT GCC TGT GTT GAT AAT TTG GCA 1742
Thr Cys Cys Thr Leu Ser Glu Glu Phe Ala Cys Val Asp Asn Leu Ala
440 445 450

GAT TTA GTT TTT GGA GAG TTA TGT GGA GTA AAT GAA AAT CGA ACT ATC 1790
Asp Leu Val Phe Gly Glu Leu Cys Gly Val Asn Glu Asn Arg Thr lie
455 460 465 470

AAC CCT GCT GTG GAC CAC TGC TGT AAA ACA AAC TTT GCC TTC AGA AGG 1838
Asn Pro Ala Val Asp His Cys Cys Lys Thr Asn Phe Ala Phe Arg Arg
475 480 485

CCC TGC TTT GAG AGT TTG AAA GCT GAT AAA ACA TAT GTG CCT CCA CCT 1886
Pro Cys Phe Glu Ser Leu Lys Ala Asp Lys Thr Tyr Val Pro Pro Pro
490 495 500

TTC TCT CAA GAT TTA TTT ACC TTT CAC GCA GAC ATG TGT CAA TCT CAG 1934 .

Phe Ser Gin Asp Leu Phe Thr Phe His Ala Asp Met Cys Gin Ser Gin
505 510 515

AAT GAG GAG CTT CAG AGG AAG ACA GAC AGG TTT CTT GTC AAC TTA GTG 1982
Asn Glu Glu Leu Gin Arg Lys Thr Asp Arg Phe Leu Val Asn Leu Val
520 525 530

AAG CTG AAG CAT GAA CTC ACA GAT GAA GAG CTG CAG TCT TTG TTT ACA 2030
Lys Leu Lys His Glu Leu Thr Asp Glu Glu Leu Gin Ser Leu Phe Thr
535 540 5415 550

AAT TTC GCA AAT GTA GTG GAT AAG TGC TGC AAA GCA GAG AGT CCT GAA 2078
Asn Phe Ala Asn Val Val Asp Lys Cys Cys Lys Ala Glu Ser Pro Glu
555 560 565

GTC TGC TTT AAT GAA GAG AGT CCA AAA ATT GGC AAC TGAAGCCAGC ' 2124

Val Cys Phe Asn Glu Glu Ser Pro Lys He Gly Asn
570 575

TGCTGGAGAT ATGTAAAGAA AAAAGCACCA AAGGGAAGGC TTCCTATCTG TGTGGTGATG 2184

AATCGCATTT CCTGAGAACA AAATAAAAGG ATTTTTCTGT AACTGTCACC TGAAATAATA 2244

CATTGCAGCA AGCAATAAAC ACAACATTTT GTAAAGTTAA AAA 2287

(2) INFORMATION FOR SEQ ID NO:2:

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PCT/US95/04075

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 599 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Leu Leu Lys Leu Thr Gly Phe He Phe Phe Leu Phe Phe Leu
-21 -20 " -15 -10

Thr Glu Ser Leu Thr Leu Pro Thr Gin Pro Arg Asp He Glu Asn Phe
-5 1 5 10

Asn Ser Thr Gin Lys Phe He Glu Asp Asn He Glu Tyr He Thr He
15 20 25

He Ala Phe Ala Gin Tyr Val Gin Glu Ala Thr Phe Glu Glu Met . Glu
30 35 40

Lys Leu Val Lys Asp Met Val Glu Tyr Lys Asp Arg Cys Met Ala Asp
45 50 55

Lys Thr Leu Pro Glu Cys Ser Lys Leu Pro Asn Asn Val Leu Gin Glu
60 65 70 75

Lys He Cys Ala Met Glu Gly Leu Pro Gin Lys His Asn Phe Ser His
80 B5 90

Cys Cys Ser Lys Val Asp Ala Gin Arg Arg Leu Cys Phe Phe Tyr Asn
95 100 105

Lys Lys Ser Asp Val Gly Phe Leu Pro Pro Phe Pro Thr Leu Asp Pro
110 115 120

Glu Glu Lys Cys Gin Ala Tyr Glu Ser Asn Arg Glu Ser Leu Leu Asn
125 130 135

His Phe Leu Tyr Glu Val Ala Arg Arg Asn Pro Phe Val Phe Ala Pro
140 145 150 155

Thr Leu Leu Thr Val Ala Val His Phe Glu Glu Val Ala Lys Ser Cys
160 165 170

Cys Glu Glu Gin Asn Lys Val Asn Cys Leu Gin Thr Arg Ala He Pro
175 180 185

Val Thr Gin Tyr Leu Lys Ala Phe Ser Ser Tyr Gin Lys His Val Cys
190 195 • 200

Gly Ala Leu Leu Lys Phe Gly Thr Lys Val Val His Phe He Tyr He
205 210 215

Ala He Leu Ser Gin Lys Phe Pro Lys lie Glu Phe Lys Glu Leu He
220 225 230 235

Ser Leu Val Glu Asp Val Ser Ser Asn Tyr Asp Gly Cys Cys Glu Gly

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240 245 250

Asp Val Val Gin Cys lie Arg Asp Thr Ser Lys Val Met Asn His lie
255 260 265

Cys Ser Lys Gin Asp Ser lie Ser Ser Lys lie Lys Glu Cys Cys Glu
270 275 280

Lys Lys lie Pro Glu Arg Gly Gin Cys lie He Asn Ser Asn Lys Asp
285 290 295

Asp Arg Pro Lys Asp Leu Ser Leu Arg Glu Gly Lys Phe Thr Asp Ser
300 305 310 315

Glu Asn Val Cys Gin Glu Arg Asp Ala Asp Pro Asp Thr Phe Phe Ala
320 325 330

Lys Phe Thr Phe Glu Tyr Ser Arg Arg His Pro Asp Leu Ser He Pro
335 340 345

Glu Leu Leu Arg He Val Gin He Tyr Lys Asp Leu Leu Arg Asn Cys
350 " 355 360

Cys Asn Thr Glu Asn Pro Pro Gly Cys Tyr Arg Tyr Ala Glu Asp Lys
365 370 375

Phe Asn Glu Thr Thr Glu Lys Ser Leu Lys Met Val Gin Gin Glu Cys
380 385 390 395

Lys His Phe Gin Asn Leu Gly Lys Asp Gly Leu Lys Tyr His Tyr Leu
400 405 410

He Arg Leu Thr Lys He Ala Pro Gin Leu Ser Thr Glu Glu Leu Val
415 420 425

Ser Leu Gly Glu Lys Met Val Thr Ala Phe Thr Thr Cys Cys Thr Leu
430 ^ 435 440

Ser Glu Glu Phe Ala Cys Val Asp Asn Leu Ala Asp Leu Val Phe Gly
445 450 455

Glu Leu Cys Gly Val Asn Glu Asn Arg Thr He Asn Pro Ala Val Asp
460 ^ 465 470 475

His Cys Cys Lys Thr Asn Phe Ala Phe Arg Arg Pro Cys Phe Glu Ser.

480 485 490

Leu Lys Ala Asp Lys Thr Tyr Val Pro Pro Pro Phe Ser Gin Asp Leu
495 500 505

Phe Thr Phe His Ala Asp Met Cys Gin Ser Gin Asn Glu Glu Leu Gin
510 515 520

Arg Lys Thr Asp Arg Phe Leu Val Asn Leu Val Lys Leu Lys His Glu
525 " ~ 530 535

Leu Thr Asp Glu Glu Leu Gin Ser Leu Phe Thr Asn Phe Ala Asn Val
540 545 550 555

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Val Asp Lys Cys Cys Lys Ala Glu Ser Pro Glu Val Cys Phe Asn Glu
560 565 570

Glu Ser Pro Lys lie Gly Asn
575

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 9 amino acids

(B) TYPE: amino acid

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Lys Trp Val Thr Phe lie Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala
20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu
35 ~ 40 45

lie Ala Phe Ala Gin Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val
50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp
65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp
85 ' 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala
100 105 110

Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin
115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val
130 135 140

Asp Val Met Cys Thr Ala Phe -His Asp Asn Glu Glu Thr Phe Leu Lys
145 150 155 160

Lys Tyr Leu Tyr Glu lie Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro
165 170 175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys
180 185 190

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Cys Gin Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu
195 200 205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gin Arg Leu Lys Cys
210 215 220

Ala Ser Leu Gin Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val
225 230 235 240

Ala Arg Leu Ser Gin Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser
245 250 255 '

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly
260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr lie
275 280 285

Cys Glu Asn Gin Asp Ser lie Ser Ser Lys Leu Lys Glu Cys Cys Glu
290 295 300

Lys Pro Leu Leu Glu Lys Ser His Cys lie Ala Glu Val Glu Asn Asp
305 310 * 315 320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser
325 330 335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly
340 345 350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val
355 360 365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys
370 375 ~ 380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu
385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro Gin Asn Leu He Lys Gin Asn Cys
405 410 415

Glu Leu Phe Lys Gin Leu Gly Glu Tyr Lys Phe Gin Asn Ala Leu Leu
420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val
435 440 445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His
450 455- 460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val
465 470 475 480

Leu Asn Gin Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg
485 490 495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

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PCT/US95/04075

500 505 510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala
515 520 525

Glu Thr Phe Thr Phe His Ala Asp lie Cys Thr Leu Ser Glu Lys Glu
530 535 540

Arg Gin lie Lys Lys Gin Thr Ala Leu Val Glu Leu Val Lys His Lys
545 550 555 560

Pro Lys Ala Thr Lys Glu Gin Leu Lys Ala Val Met Asp Asp Phe Ala
565 570 575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe
580 585 590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gin Ala Ala Leu Gly
595 600 605

Leu

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 609 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Lys Trp Val Glu Ser lie Phe Leu lie Phe Leu Leu Asn Phe Thr
15 10 15

Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly lie Ala Ser lie Leu
20 25 30

Asp Ser Tyr Gin Cys Thr Ala Glu lie Ser Leu Ala Asp Leu Ala Thr
35 40 45

lie Phe Phe Ala Gin Phe Val Gin Glu Ala Thr Tyr Lys Glu Val Ser
50 55 60

Lys Met Val Lys Asp Ala Leu Thr Ala lie Glu Lys Pro Thr Gly Asp
65 70 75 80

Glu Gin Ser Ser Gly Cys Leu Glu Asn Gin Leu Pro Ala Phe Leu Glu
85 90 95

Glu Leu Cys His Glu Lys Glu lie Leu Glu Lys Tyr Gly His Ser Asp
100 105 " ~ 110

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Cys Cys Ser Gin Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His
115 120 125

Lys Lys Pro Thr Pro Ala Ser lie Pro Leu Phe Gin Val Pro Glu Pro
130 135 140

Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn
145 ~ 150 155 160

Lys Phe lie Tyr Glu lie Ala Arg Arg His Pro Phe Leu Tyr Ala Pro
165 170 175 '

Thr lie Leu Leu Trp Ala Ala Arg Tyr Asp Lys lie lie Pro Ser Cys
180 185 190

Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gin Thr Lys Ala Ala Thr
195 200 205

Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gin His Ala Cys
210 215 220

Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gin Ala lie Thr Val
225 230 235 240

Thr Lys Leu Ser Gin Lys Phe Thr Lys Val Asn Phe Thr Glu lie Gin
245 250 255

Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly
260 265 270

Asp Val Leu Asp Cys Leu Gin Asp Gly Glu Lys lie Met Ser Tyr lie
275 280 ~ 285

Cys Ser Gin Gin Asp Thr Leu Ser Asn Lys lie Thr Glu Cys Cys Lys
290 295 300

Leu Thr Thr Leu Glu Arg Gly Gin Cys lie lie His Ala Glu Asn Asp
305 310 315 320

Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp
325 330 335

Arg Asp Phe Asn Gin Phe Ser Ser Gly Glu Lys Asn lie Phe Leu Ala
340 345 350

Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gin Leu Ala Val Ser
355 360 365

Val lie Leu Arg Val Ala Lys Gly Tyr Gin Glu Leu Leu Glu Lys Cys
370 375- 380

Phe Gin Thr Glu Asn Pro Leu Glu Cys Gin Asp Lys Gly Glu Glu Glu
385 390 395 400

Leu Gin Lys Tyr He Gin Glu Ser Gin Ala Leu Ala Lys Arg Ser Cys
405 410 415

Gly Leu Phe Gin Lys Leu Gly Glu Tyr Tyr Leu Gin Asn Ala Phe Leu

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420 425 430

Val Ala Tyr Thr Lys Lys Ala Pro Gin Leu Thr Ser Ser Glu Leu Met
435 " 440 445

Ala lie Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gin Leu
450 455 460

Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp lie lie
465 470 475 480

lie Gly His Leu Cys He Arg His Glu Met Thr Pro Val Asn Pro Gly
485 490 495

Val Gly Gin Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe
500 "* 505 510

Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp
515 520 525

Asp Lys Phe He Phe His Lys Asp Leu Cys Gin Ala Gin Gly Val Ala
530 535 540

Leu Gin Thr Met Lys Gin Glu Phe Leu He Asn Leu Val Lys Gin Lys
545 550 555 560

Pro Gin He Thr Glu Glu Gin Leu Glu Ala Val He Ala Asp Phe Ser
565 570 575

Gly Leu Leu Glu Lys Cys Cys Gin Gly Gin Glu Gin Glu Val Cys Phe
580 585 590

Ala Glu Glu Gly Gin Lys Leu He Ser Lys Thr Arg Ala Ala Leu Gly
595 * 600 605

Val

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 474 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Lys Arg Val Leu Val Leu Leu Leu Ala Val Ala Phe Gly His Ala
15 10 15

Leu Glu Arg Gly Arg Asp Tyr Glu Lys Asn Lys Val Cys Lys Glu Phe
20 25 30

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Ser His Leu Gly Lys Glu Asp Phe Thr Ser Leu Ser Leu Val Leu Tyr
35 40 45

.Ser Arg Lys Phe Pro Ser Gly Thr Phe Glu Gin Val Ser Gin Leu Val
50 55 60

Lys Glu Val Val Ser Leu Thr Glu Ala Cys Cys Ala Glu Gly Ala Asp
65 70 75 80

Pro Asp Cys Tyr Asp Thr Arg Thr Ser Ala Leu Ser Ala Lys Ser Cys
85 90 95

Glu Ser Asn Ser Pro Phe Pro Val His Pro Gly Thr Ala Glu Cys Cys
100 105 110

Thr Lys Glu Gly Leu Glu Arg Lys Leu Cys Met Ala Ala Leu Lys His
115 ~ 120 125

Gin Pro Gin Glu Phe Pro Thr Tyr Val Glu Pro Thr Asn Asp Glu lie
130 135 140

Cys Glu Ala Phe Arg Lys Asp Pro Lys Glu Tyr Ala Asn Gin Phe Met
145 150 155 160

Trp Glu Tyr Ser Thr Asn Tyr Gly Gin Ala Pro Leu Ser Leu Leu Val
165 170 175

Ser Tyr Thr Lys Ser Tyr Leu Ser Met Val Gly Ser Cys Cys Thr Ser
180 185 190

Ala Ser Pro Thr Val Cys Phe Leu Lys Glu Arg Leu Gin Leu Lys His
195 200 205

Leu Ser Leu Leu Thr Thr Leu Ser Asn Arg Val Cys Ser Gin Tyr Ala
210 215 220

Ala Tyr Gly Glu Lys Lys Ser Arg Leu Ser Asn Leu lie Lys Leu Ala
225 230 235 240

Gin Lys Val Pro Thr Ala Asp Leu Glu Asp Val Leu Pro Leu Ala Glu
245 250 255

Asp He Thr Asn He Leu Ser Lys Cys Cys Glu Ser Ala Ser Glu Asp
260 265 270

Cys Met Ala Lys Glu Leu Pro Glu His Thr Val Lys Leu Cys Asp Asn
275 280 285

Leu Ser Thr Lys Asn Ser Lys Phe Glu Asp Cys Cys Gin Glu Lys Thr
290 295 • 300

Ala Met Asp Val Phe Val Cys Thr Tyr Phe Met Pro Ala Ala Gin Leu
305 " 310 315 320

Pro Glu Leu Pro Asp Val Glu Leu Pro Thr Asn Lys Asp Val Cys Asp
325 330 335

Pro Gly Asn Thr Lys Val Met Asp Lys Tyr Thr Phe Glu Leu Ser Arg

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340 345 350

Arg Thr His Leu Pro Glu Val Phe Leu Ser Lys Val Leu Glu Pro Thr
355 360 ~ 365

Leu Lys Ser Leu Gly Glu Cys Cys Asp Val Glu Asp Ser Thr Thr Cys
370 375 380

Phe Asn Ala Lys Gly Pro Leu Leu Lys Lys Glu Leu Ser Ser Phe lie
385 " 390 395 400

Asp Lys Gly Gin Glu Leu Cys Ala Asp Tyr Ser Glu Asn Thr Phe Thr
405 410 415

Glu Tyr Lys Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu Pro Asp
420 425 430

Ala Thr Pro Lys Glu Leu Ala Lys Leu Val Asri Lys Arg Ser Asp Phe
435 440 445

Ala Ser Asn Cys Cys Ser lie Asn Ser Pro Pro Leu Tyr Cys Asp Ser
450 455 460

Glu He Asp Ala Glu Leu Lys Asn He Leu
465 470

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Leu Pro Thr Gin Pro Arg Asp He Glu Asn Phe Xaa Ser Thr Gin Lys
15 10 15

Phe He Glu Asp Asn He Glu Tyr He Thr He He Ala Phe Ala Gin
20 25 30

Tyr Val Gin
35

(2) INFORMATION FOR SEQ ID NO: 7:-

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

WO 95/27059 PCTYUS95/04075

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Tyr His Tyr Leu lie Arg
1 5

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

<ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Phe Thr Phe Glu Tyr Ser Arg
1 5

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Phe Thr Asp Ser Glu Asn Val Cys Gin Glu Arg Asp Ala Asp Pro
1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

lie Val Gin lie Tyr Lys Asp Leu Leu Arg
1 5 10

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PCT/US95/04075

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

lie Ala Pro Gin Leu Ser Thr Glu Glu Leu Val Ser Leu Gly Glu
15 10 15

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Arg His Pro Asp Leu Ser lie Pro Glu Leu Leu Arg
1 5 10

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Glu Ser Leu Leu Asn His Phe- Leu Tyr Glu Val Ala Arg
1 5 10

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid

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(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Arg Asn Pro Phe Val Phe Ala Pro Thr Leu Leu Thr Val Ala Val His
1 5 10 15

Phe Glu Glu Val Ala Lys Ser Cys Cys
20 25

(2) INFORMATION FOR SEQ ID NO: 15:

<i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE .TYPE : peptide

<xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Gin Lys Phe lie Glu Asp Asn
1 5

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:*

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
ACGCTGAATT CGCCARAART TYATHGARGA YAA 33
(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

WO 95/27059

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17

. lie Val Gin lie Tyr Lys Asp
1 5

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18
ACGCTAAGCT TGCRTCYTTR TADATYTGNA CDAT
(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19

Asp Asn lie Glu Tyr He Thr
1 5

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20
ACGCTGAATT CGCGAYAAYA THGARTAYAT HAC
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:

WO 95/27059

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21

Phe Thr Phe Glu Tyr Ser
1 5

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE . TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22

ACGCTAAGCT TGCNGARTAY TCRAANGTRA A

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:
<A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

( C ) S TRANDEDNES S : unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23
TATGTGCTAT GGAGGGGC
(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

WO 95/27059

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24
CAACCCTGCT GTGGACCA
(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25
GCACATATGT TTTATCAGCT TT
(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26

TCACCTCTAG ACCACCATGA AACTACTAAA ACTTACAG

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(6) TYPE : nucleic acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27
AATTTCTCAG GAGATCTTTG TATA
(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

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(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
AAATATACAA AGATCTCCTG AGAA 24
(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: unknown

<ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
TCCCGGTCGA CTCAGTTGCC AATTTTTGGA C 31
(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Asp Leu Ser Leu Arg Glu Gly Lys Phe Thr Asp Ser Glu Asn Val Cys
1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

( C ) S TRANDEDNES S : unknown

(D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

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Glu Lys Leu Val Lys Asp Met Val Glu Tyr Lys Asp Arg Cys
1 5 10

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Cys lie lie Asn Ser Asn Lys Asp Asp Arg Pro Lys Asp Leu Ser Leu
1 5 10 15

Arg

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Cys Gin Glu Arg Asp Ala Asp Pro Asp Thr Phe Phe Ala Lys Phe Thr
15 10 15

WO 95/27059

PCT/DS95/04075

The embodiments of the invention in which an
exclusive property or privilege is claimed are defined as
follows :

5 1. A purified and isolated polynucleotide

encoding AFM polypeptide or a variant thereof possessing a
biological activity specific to AFM.

2 . The polynucleotide of claim 1 which is a
1 0 DNA sequence .

3. The DNA sequence according to claim 2 which
is a cDNA sequence or a biological replica thereof.

15 4. The cDNA sequence of claim 3, which encodes

AFM.

5. The DNA sequence according to claim 3 which
is a genomic DNA sequence or a biological replica thereof.

6. The DNA se.quence of claim 3 further
including an endogenous expression control DNA sequence.

7 . The DNA sequence according to claim 3 which
2 5 is a wholly or partially chemically synthesized DNA

sequence or a biological replica thereof.

8. A DNA vector comprising a DNA sequence
according to claim 3 .

9. The vector of claim 8 wherein said DNA
sequence is operatively linked to an expression control DNA
sequence .

WO 95/27059

PCTYUS95/04075

10. A host cell stably transformed or
•transfected with a DNA sequence according to claim 3 in a
manner allowing the expression in said host cell of AFM

5 polypeptide or a variant thereof possessing a biological
activity specific to AFM.

11. A method for producing AFM polypeptide or a
variant thereof possessing a biological activity specific

10 to AFM, said method comprising growing a host cell

according to claim 8 in a suitable nutrient medium and
isolating AFM polypeptide or variant thereof from said cell
or the growth medium.

15 12. Purified and isolated AFM polypeptide or a

variant thereof possessing a biological activity specific
to AFM.

13. The polypeptide of claim 12 comprising
20 amino acid residues 1 through 578 of FIG 2.

14 . The polypeptide of claim 12 comprising
residues -23 through 578 of FIG 2.

25 15. Purified and isolated AFM polypeptide

complexed with Apolipoprotein Al.

16. An antibody specific for AFM or a variant

thereof.

17. A monoclonal antibody according to claim

16.

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18. A humanized antibody according to claims 16

or 17.

19. A method for modulating a biological

5 activity of AFM comprising contacting AFM with an antibody
according to claim 16 or 17.

20. A method for detecting the capacity of a
cell to synthesize AFM comprising hybridizing detectable

1 0 polynucleotide encoding AFM or a fragment thereof with RNA
of said cell.

21. A method for detecting the capacity of a
cell to synthesize AFM comprising reacting an antibody

15 according to claim 16 with polypeptides produced by said
cell.

22. A method for determining the presence of
AFM in a sample body fluid, comprising contacting the body ■

20 fluid with an antibody according to Claim 16.

23. An antisense polynucleotide for a
polynucleotide encoding AFM.

25 24. An antisense polynucleotide for a DNA

specifying an endogenous expression control DNA sequence of
AFM.

25. A hybrid fusion polypeptide comprising, at
3 0 its amino terminal, an AFM polypeptide or a variant thereof
possessing a biological- activity specific to AFM and, at
its carboxy terminal f at least one constant domain of an
immunoglobulin heavy chain or allelic variant thereof.

WO 95/27059

PCT/US95/04075

26. A polynucleotide encoding a hybrid fusion
protein according to claim 25.

5 27. A DNA sequence encoding a polypeptide

having a biological property specific for AFM and selected
from the group consisting of :

(a) the DNA sequence set out in FIG 1;

(b) a DNA which hybridizes under stringent
0 conditions to the DNA of (a) ; and

(c) a DNA sequence which, but for the
redundancy of the genetic code, would hybridize under
stringent conditions to a DNA sequence of (a) or (b) .

WO 95/27059 PCT/US95/04075

1/12

FIG. IA

CCCCGAGTCTCTGCGCCTTCACATAGTTGTCACAGGACTAAAGCAAATTGATCCAGGGGG 6 0

AAACACTGTAGACCGTGTATATAAAAACACTCTATAAACTGCAATGCTCAATTCTTAGTA 12 0

TAACTATTGTTGTTGTATTGATATTTATTAGTATTGGTGCTCACAAAAAGAGTCTAAATT 18 0

CCATAAGTCTTTATATTC AGGCTACTCTTTATTTTTGAAAACTC ATTTTCTATC ACCTTT 240

TTCTATTTTACTCCATATTGAGGCCTCATAAATCCAATTTTTTATTTCTTTCTTTTGTAA 300

m k 1 1 k 1 t g f i f f 1 f - f -21

ATGTGGTTTCTACAAAGATGAAACTACTAAAACTTACAGGTTTTATTTTTTTCTTGTTTT 360

ItesltLPTQPRDIENFNST 14

TTTTGACTGAATCCCTAACCCTGCCCACACAACCTCGGGATATAGAGAACTTCAATAGTA 420

QKFIEDNIEYITIIAFAQYV 34

CTCAAAAATTTATAGAAGATAATATTGAATACATCACCATCATTGC ATTTGCTCAGTATG 480

QEAT FEEMEKLVKDMVEYKD 54

TTCAGGAAGCAACCTTTGAAGAAATGGAAAAGCTGGTGAAAGACATGGTAGAATACAAAG 540

RCMADKTLPECSKLPNNVLQ 74

ACAGATGTATGGCTGACAAGACGCTCCCAGAGTGTTCAAAATTACCTAATAATGTTTTAC 600

EKICAM.EGLPQKHNFSHCCS 94

AGGAAAAAATATGTGCTATGGAGGGGCTGCCACAAAAGCATAATTTCTCACACTGCTGCA 660

KVDAQRRLCFFYNKKSDVGF 114

GTAAGGTTGATGCTCAAAGAAGACTCTGTTTCTTCTATAACAAGAAATCTGATGTGGGAT 720

LPPFPTLDPEEKCQAYESNR 134

TTCTGCCTCCTTTCCCTACCCTGGATCCCGAAGAGAAATGCCAGGCTTATGAAAGTAACA 780

ESLLNHFLYEVARRNPFVFA 154

GAGAATCCCTTTTAAATCACTTTTTATATGAAGTTGCCAGAAGGAACCCATTTGTCTTCG 840

PTLLTVAVHFEEV AKSCCEE 174

CCCCTACACTTCTAACTGTTGCTGTTCATTTTGAGGAGGTGGCCAAATCATGTTGTGAAG 900

QNKVNCLQTRAIPVTQYLKA 194

AACAAAACAAAGTCAACTGCCTTCAAACAAGGGCAATACCTGTCACACAATATTTAAAAG 960

FS SYQKHVC GALLKFGTKVV 214

CATTTTCTTCTTATCAAAAACATGTCTGTGGGGCACTTTTGAAATTTGGAACCAAAGTTG 1020

HFIYIAILSQKFPKIEFKEL 234

TACACTTTATATATATTGCGATACTCAGTCAAAAATTCCCCAAGATTGAATTTAAGGAGC 1080

ISLVEDVSSNYDGCCEGDVV 254

TTATTTCTCTTGTAGAAGATGTTTCTTCCAACTATGATGGATGCTGTGAAGGGGATGTTG 1140

PCT/US95/04075

2/12

FIG. IB

QC I RDTSKVMNH IC SKQDS I 274

TGCAGTGCATCCGTGACACGAGCAAGGTTATGAACCATATTTGTTCAAAACAAGATTCTA 1200

SSKIKECCEKKIPERGQCII 294

TCTCCAGCAAAATCAAAGAGTGCTGTGAAAAGAAAATACCAGAGCGCGGCCAGTGCATAA 1260

NSNKDDRPKDLSLREGKFTD 314

TTAACTCAAACAAAGATGATAGACCAAAGGATTTATCTCTAAGAGAAGGAAAATTTACTG 1320

SE NVCQERDADP DTFFAKFT 334

ACAGTGAAAATGTGTGTCAAGAACGAGATGCTGACCCAGACACCTTCTTTGCGAAGTTTA 1380

FEYSRRH PDLSIPELLRIVQ 354

CTTTTGAATACTCAAGGAGACATCCAGACCTGTCTATACCAGAGCTTTTAAGAATTGTTC 1440

IYKDLLRNCCNTENPPGCYR 374

AAATATACAAAGATCTCCTGAGAAATTGCTGCMCACAGAAAACCCTCCAGGTTGTTACC 1500

YAEDKFNETTEKSLKMVQQE 394

GTTACGCGGAAGACAAATTCAATGAGACAACTGAGAAAAGCCTCAAGATGGTACAACAAG 1560

CKHFQNLGKDGLKYH YLIRL 414

AATGTAAACATTTCCAGAATTTGGGGAAGGATGGTTTGAAATACCATTACCTCATCAGGC 1 62 0

TKIAPQ LSTEELVSLGEKMV 434

TCACGAAGATAGCTCCCCAACTCTCCACTGAAGAACTGGTGTCTCTTGGCGAGAAAATGG 1680

TAFTTCCTLSEEFACVDNLA 454

TGACAGCTTTCACTACTTGCTGTACGCTAAGTGAAGAGTTTGCCTGTGTTGATAATTTGG 1740

DLVFGELCGVNENRTINPAV 474

CAGATTTAGTTTTTGGAGAGTTATGTGGAGTAAATGAAAATCGAACTATCAACCCTGCTG 1800

DHCCKTNFAFRRPCFESLKA 494

TGGACCACTGCTGTAAAACAAACTTTGCCTTCAGAAGGCCCTGCTTTGAGAGTTTGAAAG 1860

DKTYVPPPFSQDLFTFHADM 514

CTGATAAAACATATGTGCCTCCACCTTTCTCTCAAGATTTATTTACCTTTCACGCAGACA 1920

CQSQNEELQRKTDRFLVNLV 534

TGTGTCAATCTCAGAATGAGGAGCTTCAGAGGAAGACAGACAGGTTTCTTGTCAACTTAG 1980

KLKHEL.TDEELQSLFTNFAN 554

TGAAGCTGAAGCATGAACTCACAGATGAAGAGCTGCAGTCTTTGTTTACAAATTTCGCAA 2040

VVDKCCKAESP EVCFNEESP 574

ATGTAGTGGATAAGTGCTGCAAAGCAGAGAGTCCTGAAGTCTGCTTTAATGAAGAGAGTC 2100

SUBSTITUTE SHEET (RULE 3S)

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FIG. IC

K I G N J>H

CAAAAATTGGCAACTGAAGCCAGCTGCTGGAGATATGTAAAGAAAAAAGCACCAAAGGGA 2160

AGGCTTCCTATCTGTGTGGTGATGAATCGCATTTCCTGAGAACAAAATAAAAGGATTTTT 2220

CTGTAACTGTCACCTGAAATAATACATTGCAGCAAGCAATAAACACAACATTTTGTAAAG 2280

TTAAAAA 2287

eiiBCTrrinr fsUFfT fRUlE 28)

WO 95/27059

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PCT/US95/04075

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FIG. 2B

% Similarity

AFM AFP ALB VDB

siipmrrm: aim im F 2ft

wo 95/27059

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PCTAJS95/04075

(

WO 95/27059

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PCT7US95/04075

AimmiifV AiirrT /mil f Oj^a

PCT/US 95/04075

A.' CLASSIFICATION OF SUBJECT MATTER . ... „ A - 1/ - - /1Q nmUOO/CS

IPC 6 C12N15/12 C12N15/62 C07K14/47 C07K16/18 G01N33/53

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 6 C07K

Documentation searched other than minimum documentation to the extent that such documents arc included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

THE JOURNAL OF BIOLOGICAL CHEMISTRY,
vol. 269, no. 8, 25 February 1994
pages 5481-5484,

B6LANGER ET AL 'NEW ALBUMIN GENE 3'
ADJACENT TO THE ALPHA1-FET0PR0TEIN LOCUS'
see the whole document

THE JOURNAL OF BIOLOGICAL CHEMISTRY,
vol. 269, no. 27, 8 July 1994
pages 18149-18154,

LICHENSTEIN ET AL 'AFAMIN IS A NEW MEMBER
OF THE ALBUMIN, ALPHA-FETOPROTEIN, AND
VITAMIN D-BINDING PROTEIN GENE FAMILY'
see the whole document

-/~

1-27

Further documents arc listed in the continuation of box C.

Patent family members are listed in annex.

* Special categories of cited documents :

# A' document defining the general state of the art which is not

considered to be of particular relevance
*E* earlier document but published on or after the international

filing date

*L* document which may throw doubts on priority daim(s) or •
which is cited to establish the publication date of another
citation or other special reason (as specified)

*0 # document referring to an oral disclosure, use, exhibition or
other means

# P* document published prior to the international filing date but
later the priority date claimed

*T* later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention

'X' document of particular relevance; the claimed invention
cann ot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone

* Y* document of particular relevance; the daimed invention
cann ot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art

*&* document member of the same patent family

Date of the actual completion of the international search

2 August 1995

Date of mailing of the international search report

1 0. 08. 95

Name and mailing address of the ISA

European Patent Office, P.B. 581 8 Patenuaan 2
NL * 2280 HV Rijswijk
Td. (+ 31-70) 340-2040, TX. 31 651 epo nl,
Fax ( + 31-70) 34O-3016

Authorized officer

Sitch> W

Form PCT/ISA/310 (i

id ihMt) (July 1993)

{ PCT/US 95/04075

C^Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

WO, A, 95 11308 (AMGEN INC) 27 April 1995

see page 6, line 20 - page 35

see SEQ ID NOS: 23 and 24

see page 41 - page 43; example 4

1-H.16,
27

Form PCT/ISA/310 (continumtioo of ucond theet) (July 1993)

Patent document
cited in search report

Publication
date

PCT/US 95/04075

Patent family
member(s)

Publication
date

WO-A-9511308

27-04-95

AU-B- 7979194

08-05-95

Form PCT/ISA/310 (paUM family annex) (July 1993)

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